Compositions and methods for ophthalmic and/or other applications

ABSTRACT

Particles, compositions, and methods that aid particle transport in mucus are provided. The particles, compositions, and methods may be used, in some instances, for ophthalmic and/or other applications. In some embodiments, the compositions and methods may involve modifying the surface coatings of particles, such as particles of pharmaceutical agents that have a low aqueous solubility. Such compositions and methods can be used to achieve efficient transport of particles of pharmaceutical agents though mucus barriers in the body for a wide spectrum of applications, including drug delivery, imaging, and diagnostic applications. In certain embodiments, a pharmaceutical composition including such particles is well-suited for ophthalmic applications, and may be used for delivering pharmaceutical agents to the front of the eye and/or the back of the eye.

RELATED APPLICATIONS

The present application is a division of U.S. application Ser. No.15/808,746 filed Nov. 9, 2017, which is a continuation of U.S.application Ser. No. 14/070,506 filed Nov. 2, 2013, which is acontinuation-in-part of U.S. application Ser. No. 13/886,658 filed May3, 2013, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Applications 61/784,701 filed Mar. 14, 2013, 61/642,313filed May 3, 2012, 61/642,261 filed May 3, 2012, and 61/738,949 filedDec. 18, 2012. Each of these applications is incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made in part with government support under Grant No.1R43EY021705-1 awarded by the National Eye Institute of the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention generally relates to particles, compositions, andmethods that aid particle transport in mucus. The particles,compositions, and methods may be used in ophthalmic and/or otherapplications.

BACKGROUND OF THE INVENTION

A mucus layer present at various points of entry into the body,including the eyes, nose, lungs, gastrointestinal tract, and femalereproductive tract, is naturally adhesive and serves to protect the bodyagainst pathogens, allergens, and debris by effectively trapping andquickly removing them via mucus turnover. For effective delivery oftherapeutic, diagnostic, or imaging particles via mucus membranes, theparticles must be able to readily penetrate the mucus layer to avoidmucus adhesion and rapid mucus clearance. Particles (includingmicroparticles and nanoparticles) that incorporate pharmaceutical agentsare particularly useful for ophthalmic applications. However, often itis difficult for administered particles to be delivered to an eye tissuein effective amounts due to rapid clearance and/or other reasons.Accordingly, new methods and compositions for administration (e.g.,topical application or direct injection) of pharmaceutical agents to theeye would be beneficial.

SUMMARY OF THE INVENTION

The present description generally relates to particles, compositions,and methods that aid particle transport in mucus, especially particles,compositions, and methods for ophthalmic and/or other applications.

As described in more detail below, in some embodiments the compositionscomprise a plurality of particles that include a corticosteroid such asloteprednol etabonate (LE) for treating an eye disease or condition. Theparticles include a surface-altering agent that reduces the adhesion ofthe particles to mucus and/or facilitates penetration of the particlesthrough physiological mucus. Such compositions are advantageous overmarketed formulations, such as Lotemax® or Alrex®, as the compositionsdescribed herein are able to more readily penetrate the mucus layer ofan ocular tissue to avoid or minimize mucus adhesion and/or rapid mucusclearance. Therefore, the compositions may be more effectively deliveredto and may be retained longer in the target issue. As a result, thecompositions described herein may be administered at a lower dose and/orless frequently than marketed formulations to achieve similar orsuperior exposure. Moreover, the relatively low and/or infrequent dosageof the compositions described herein may result in fewer or less severeside effects, a more desirable toxicity profile, and/or improved patientcompliance.

In some embodiments, the compositions described herein may comprise aplurality of particles that include a receptor tyrosine kinase (RTK)inhibitor, such as sorafenib, linifanib, MGCD-265, pazopanib, cediranib,and axitinib, for treating an eye disease or condition. Compositionsincluding such particles are also provided, including compositions thatcan be administered topically to the eye. For reasons described herein,such compositions may have certain advantages over conventionalformulations (e.g., an aqueous suspension of such pharmaceuticalagents).

In certain embodiments, the compositions described herein may comprise aplurality of particles that include a non-steroidal anti-inflammatorydrug (NSAID), such as a divalent or trivalent metal salt of bromfenac(e.g., bromfenac calcium), diclofenac (e.g., diclofenac free acid or adivalent or trivalent metal salt thereof), or ketorolac (e.g., ketorolacfree acid or a divalent or trivalent metal salt thereof), for treatingan eye disease or condition. Compositions including such particles arealso provided, including compositions that can be administered topicallyto the eye. For reasons described herein, such compositions may haveadvantages over conventional formulations (e.g., an aqueous solution ofthe corresponding pharmaceutical agent).

In one set of embodiments, a pharmaceutical composition suitable foradministration to an eye is provided. The pharmaceutical compositioncomprises a plurality of coated particles, comprising a core particlecomprising loteprednol etabonate, wherein the loteprednol etabonateconstitutes at least about 80 wt % of the core particle, and a coatingcomprising one or more surface-altering agents surrounding the coreparticle. The one or more surface-altering agents comprises at least oneof: a) a triblock copolymer comprising a hydrophilic block-hydrophobicblock-hydrophilic block configuration, wherein the hydrophobic block hasa molecular weight of at least about 2 kDa, and the hydrophilic blocksconstitute at least about 15 wt % of the triblock copolymer, b) asynthetic polymer having pendant hydroxyl groups on the backbone of thepolymer, the polymer having a molecular weight of at least about 1 kDaand less than or equal to about 1000 kDa, wherein the polymer is atleast about 30% hydrolyzed and less than about 95% hydrolyzed, or c) apolysorbate. The one or more surface altering agents is present on theouter surface of the core particle at a density of at least 0.01molecules/nm². The one or more surface altering agents is present in thepharmaceutical composition in an amount of between about 0.001% to about5% by weight. The plurality of coated particles have an average smallestcross-sectional dimension of less than about 1 micron. Thepharmaceutical composition also includes one or more ophthalmicallyacceptable carriers, additives, and/or diluents.

In another set of embodiments, a pharmaceutical composition suitable fortopical administration to an eye is provided. The pharmaceuticalcomposition comprises a plurality of coated particles, comprising a coreparticle comprising loteprednol etabonate, wherein the loteprednoletabonate constitutes at least about 80 wt % of the core particle, and acoating comprising one or more surface-altering agents, wherein the oneor more surface-altering agents comprise at least one of a poloxamer, apoly(vinyl alcohol), or a polysorbate. The one or more surface-alteringagents is present on the outer surface of the core particle at a densityof at least 0.01 molecules/nm². The one or more surface-altering agentsis present in the pharmaceutical composition in an amount of betweenabout 0.001% to about 5% by weight. The plurality of coated particleshave an average smallest cross-sectional dimension of less than about 1micron. The pharmaceutical composition also includes one or moreophthalmically acceptable carriers, additives, and/or diluents.

In another set of embodiments, a pharmaceutical composition suitable foradministration to an eye is provided. The pharmaceutical compositioncomprises a plurality of coated particles, comprising a core particlecomprising loteprednol etabonate; and a coating comprising asurface-altering agent surrounding the core particle, wherein thesurface-altering agent comprises a (poly(ethyleneoxide))-(poly(propylene oxide))-(poly(ethylene oxide)) triblockcopolymer, wherein the hydrophobic block has a molecular weight of about3600 Da, and the hydrophilic blocks constitute about 70 wt % of thetriblock copolymer. The coated particles may have an average smallestcross-sectional dimension of about 200 nm to about 500 nm or about 200nm to about 300 nm. The pharmaceutical composition may also comprise oneor more ophthalmically acceptable carriers, additives, and/or diluents.The loteprednol etabonate is present in the pharmaceutical compositionin an amount between about 0.1% to about 2% loteprednol etabonate byweight. The one or more surface altering agents is present in thepharmaceutical composition in an amount of between about 0.01% to about2% by weight. In various embodiments, the ophthalmically acceptablecarriers, additives, and/or diluents may comprise about 0.5-3% glycerin,about 0.1-1% sodium chloride, about 0.001-0.1% disodiumethylenediaminetetraacetic acid, and about 0.001-0.05% benzalkoniumchloride. In addition, the composition may further comprise sodiumcitrate, citric acid, and water. In some embodiments, the ratio of theweight of the loteprednol etabonate to the weight of thesurface-altering agent present in the pharmaceutical composition isgreater than or equal to about 1:1, and less than or equal to about 3:1.The surface-altering agent may be Pluronic® F127, and may benon-covalently adsorbed to the core particles.

In another set of embodiments, a pharmaceutical composition suitable foradministration to an eye is provided. The pharmaceutical compositioncomprises a plurality of coated particles, comprising a core particlecomprising loteprednol etabonate; and a coating comprising asurface-altering agent surrounding the core particle, wherein thesurface-altering agent comprises a (poly(ethyleneoxide))-(poly(propylene oxide))-(poly(ethylene oxide)) triblockcopolymer, wherein the hydrophobic block has a molecular weight of about3600 Da, and the hydrophilic blocks constitute about 70 wt % of thetriblock copolymer, about 0.5% to about 1% glycerin, about 0.1% to about1% sodium chloride, about 0.01% to about 0.1% disodiumethylenediaminetetraacetic acid, and about 0.01% to about 0.03%benzalkonium chloride, wherein the loteprednol etabonate is present inthe pharmaceutical composition in an amount between about 0.1% to about1% loteprednol etabonate by weight; and wherein the ratio of the weightof the loteprednol etabonate to the weight of the surface-altering agentis about 2:1. The pharmaceutical composition may also include sodiumcitrate, citric acid, and water. In some embodiments, the loteprednoletabonate is present in the pharmaceutical composition in an amount ofabout 0.25% loteprednol etabonate by weight. In other embodiments, theloteprednol etabonate is present in the pharmaceutical composition in anamount of about 1% loteprednol etabonate by weight. In some embodiments,the glycerin is present in an amount of about 0.6%.

In another set of embodiments, a series of methods are provided. In oneembodiment, a method for treating inflammation, macular degeneration,macular edema, uveitis, dry eye, blepharitis, and/or other disorder inan eye of a patient is provided. The method comprises administering toan eye of the patient, a pharmaceutical composition comprising aplurality of coated particles. The plurality of coated particlescomprise a core particle comprising loteprednol etabonate, wherein theloteprednol etabonate constitutes at least about 80 wt % of the coreparticle, and a coating comprising one or more surface-altering agentssurrounding the core particle. The one or more surface-altering agentscomprises at least one of: a) a triblock copolymer comprising ahydrophilic block-hydrophobic block-hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least about 2kDa, and the hydrophilic blocks constitute at least about 15 wt % of thetriblock copolymer, b) a synthetic polymer having pendant hydroxylgroups on the backbone of the polymer, the polymer having a molecularweight of at least about 1 kDa and less than or equal to about 1000 kDa,wherein the polymer is at least about 30% hydrolyzed and less than about95% hydrolyzed, or c) a polysorbate. The one or more surface alteringagents is present on the outer surface of the core particle at a densityof at least 0.01 molecules/nm². The one or more surface altering agentsis present in the pharmaceutical composition in an amount of betweenabout 0.001% to about 5% by weight. The plurality of coated particleshave an average smallest cross-sectional dimension of less than about 1micron. The pharmaceutical composition also includes one or moreophthalmically acceptable carriers, additives, and/or diluents.

In another set of embodiments, a method for treating inflammation,macular degeneration, macular edema, uveitis, dry eye, blepharitis,and/or other disorder in an eye of a patient is provided. The methodinvolves administering to an eye of the patient, a pharmaceuticalcomposition comprising a plurality of coated particles, comprising acore particle comprising loteprednol etabonate, wherein the loteprednoletabonate constitutes at least about 80 wt % of the core particle, and acoating comprising one or more surface-altering agents, wherein the oneor more surface-altering agents comprise at least one of a poloxamer,poly(vinyl alcohol), or a polysorbate. The one or more surface-alteringagents is present on the outer surface of the core particle at a densityof at least 0.01 molecules/nm². The one or more surface-altering agentsis present in the pharmaceutical composition in an amount of betweenabout 0.001% to about 5% by weight. The plurality of coated particleshave an average smallest cross-sectional dimension of less than about 1micron. The pharmaceutical composition also includes one or moreophthalmically acceptable carriers, additives, and/or diluents.

In another set of embodiments, a method for treating inflammation,macular degeneration, macular edema, uveitis, dry eye, blepharitis,and/or other disorder in an eye of a patient is provided. The methodinvolves administering to an eye of the patient a pharmaceuticalcomposition comprising a plurality of coated particles. The plurality ofcoated particles, comprises a core particle comprising loteprednoletabonate; and a coating comprising a surface-altering agent surroundingthe core particle, wherein the surface-altering agent comprises a(poly(ethylene oxide))-(poly(propylene oxide))-(poly(ethylene oxide))triblock copolymer, wherein the hydrophobic block has a molecular weightof about 3600 Da, and the hydrophilic blocks constitute about 70 wt % ofthe triblock copolymer. The coated particles may have an averagesmallest cross-sectional dimension of about 200 nm to about 500 nm orabout 200 nm to about 300 nm. The pharmaceutical composition can alsocomprise one or more ophthalmically acceptable carriers, additives,and/or diluents. The loteprednol etabonate is present in thepharmaceutical composition in an amount between about 0.1% to about 2%loteprednol etabonate by weight. The one or more surface altering agentsis present in the pharmaceutical composition in an amount of betweenabout 0.01% to about 2% by weight. In various embodiments, theophthalmically acceptable carriers, additives, and/or diluents maycomprise about 0.5-3% glycerin, about 0.1-1% sodium chloride, about0.001-0.1% disodium ethylenediaminetetraacetic acid, and about0.001-0.05% benzalkonium chloride. In addition, the composition may alsocomprise sodium citrate, citric acid, and water. In some embodiments,the ratio of the weight of the loteprednol etabonate to the weight ofthe surface-altering agent present in the pharmaceutical composition isgreater than or equal to about 1:1, and less than or equal to about 3:1.The surface-altering agent may be Pluronic® F127, and may benon-covalently adsorbed to the core particles.

In another set of embodiments, a method for treating inflammation,macular degeneration, macular edema, uveitis, dry eye, blepharitis,and/or other disorder in an eye of a patient is provided. The methodinvolves administering to an eye of the patient a pharmaceuticalcomposition that comprises a plurality of coated particles comprising acore particle comprising loteprednol etabonate; and a coating comprisinga surface-altering agent surrounding the core particle, wherein thesurface-altering agent comprises a (poly(ethyleneoxide))-(poly(propylene oxide))-(poly(ethylene oxide)) triblockcopolymer, wherein the hydrophobic block has a molecular weight of about3600 Da, and the hydrophilic blocks constitute about 70 wt % of thetriblock copolymer, about 0.5% to about 1% glycerin, about 0.1% to about1% sodium chloride, about 0.01% to about 0.1% disodiumethylenediaminetetraacetic acid, and about 0.01% to about 0.03%benzalkonium chloride, wherein the loteprednol etabonate is present inthe pharmaceutical composition in an amount between about 0.1% to about1% loteprednol etabonate by weight; and wherein the ratio of the weightof the loteprednol etabonate to the weight of the surface-altering agentis about 2:1. The pharmaceutical composition can also include sodiumcitrate, citric acid, and water. In some embodiments, the loteprednoletabonate is present in the pharmaceutical composition in an amount ofabout 0.25% loteprednol etabonate by weight. In other embodiments, heloteprednol etabonate is present in the pharmaceutical composition in anamount of about 1% loteprednol etabonate by weight. In some embodiments,the glycerin is present in an amount of about 0.6%.

In another set of embodiments, a method of making a compositioncomprising a plurality of coated particles comprising a core particlecomprising loteprednol etabonate; and a coating comprising asurface-altering agent surrounding the core particle, wherein thesurface-altering agent comprises a (poly(ethyleneoxide))-(poly(propylene oxide))-(poly(ethylene oxide)) triblockcopolymer, wherein the hydrophobic block has a molecular weight of about3600 Da, and the hydrophilic blocks constitute about 70 wt % of thetriblock copolymer, and one or more ophthalmically acceptable carriers,additives, and/or diluents, wherein the loteprednol etabonate is presentin the pharmaceutical composition in an amount between about 0.1% toabout 2% loteprednol etabonate by weight; wherein the one or moresurface altering agents is present in the pharmaceutical composition inan amount of between about 0.01% to about 2% by weight, wherein theplurality of coated particles have an average smallest cross-sectionaldimension of about 0.2 microns to about 0.3 microns is provided. Themethod involves milling a coarse aqueous suspension containing about2-20% loteprednol etabonate in the form of coarse or micronizedcrystals, about 0.2-20% of a (poly(ethylene oxide))-(poly(propyleneoxide))-(poly(ethylene oxide)) triblock copolymer, wherein thehydrophobic block has a molecular weight of about 3600 Da, and thehydrophilic blocks constitute about 70 wt % of the triblock copolymer,about 0.5-3% glycerin, about 0.1-1% sodium chloride, and about0.001-0.1% EDTA in the presence of milling media to produce ananosuspension of loteprednol etabonate particles sized in the range ofabout 0.2 microns to about 0.3 microns, separating the nanosuspension ofloteprednol etabonate particles from the milling media, and mixing thenanosuspension of loteprednol etabonate particles with diluent.

In another set of embodiments, a pharmaceutical composition suitable foradministration to an eye is provided. The pharmaceutical compositionincludes a plurality of coated particles, comprising a core particlecomprising a pharmaceutical agent or a salt thereof. The pharmaceuticalagent or salt thereof constitutes at least about 80 wt % of the coreparticle, and the pharmaceutical agent or salt thereof comprises areceptor tyrosine kinase (RTK) inhibitor. The plurality of coatedparticles also includes a coating comprising one or moresurface-altering agents surrounding the core particle, wherein the oneor more surface-altering agents comprises at least one of: a) a triblockcopolymer comprising a hydrophilic block-hydrophobic block-hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, b) a synthetic polymerhaving pendant hydroxyl groups on the backbone of the polymer, thepolymer having a molecular weight of at least about 1 kDa and less thanor equal to about 1000 kDa, wherein the polymer is at least about 30%hydrolyzed and less than about 95% hydrolyzed, or c) a polysorbate. Theone or more surface altering agents is present on the outer surface ofthe core particle at a density of at least 0.01 molecules/nm². The oneor more surface altering agents is present in the pharmaceuticalcomposition in an amount of between about 0.001% to about 5% by weight.The plurality of coated particles have an average smallestcross-sectional dimension of less than about 1 micron. Thepharmaceutical composition also includes one or more ophthalmicallyacceptable carriers, additives, and/or diluents.

In another set of embodiments, a method for treating maculardegeneration, macular edema and/or another disorder in an eye of apatient is provided. The method involves administering to an eye of thepatient, a pharmaceutical composition comprising a plurality of coatedparticles, comprising a core particle comprising a pharmaceutical agentor a salt thereof, wherein the pharmaceutical agent or salt thereofconstitutes at least about 80 wt % of the core particle, and wherein thepharmaceutical agent or salt thereof comprises a receptor tyrosinekinase (RTK) inhibitor, and a coating comprising one or moresurface-altering agents surrounding the core particle. The one or moresurface-altering agents comprises at least one of: a) a triblockcopolymer comprising a hydrophilic block-hydrophobic block-hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, b) a synthetic polymerhaving pendant hydroxyl groups on the backbone of the polymer, thepolymer having a molecular weight of at least about 1 kDa and less thanor equal to about 1000 kDa, wherein the polymer is at least about 30%hydrolyzed and less than about 95% hydrolyzed, or c) a polysorbate. Theone or more surface altering agents is present on the outer surface ofthe core particle at a density of at least 0.01 molecules/nm². The oneor more surface altering agents is present in the pharmaceuticalcomposition in an amount of between about 0.001% to about 5% by weight.The plurality of coated particles have an average smallestcross-sectional dimension of less than about 1 micron. Thepharmaceutical composition also includes one or more ophthalmicallyacceptable carriers, additives, and/or diluents.

In another set of embodiments, a pharmaceutical composition suitable foradministration to an eye is provided. The pharmaceutical compositioncomprises a plurality of coated particles, comprising a core particlecomprising a pharmaceutical agent or a salt thereof, wherein thepharmaceutical agent or salt thereof constitutes at least about 80 wt %of the core particle, and wherein the pharmaceutical agent or saltthereof comprises bromfenac calcium, diclofenac free acid, or ketorolacfree acid, and a coating comprising one or more surface-altering agentssurrounding the core particle. The one or more surface-altering agentscomprises at least one of: a) a triblock copolymer comprising ahydrophilic block-hydrophobic block-hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least about 2kDa, and the hydrophilic blocks constitute at least about 15 wt % of thetriblock copolymer, b) a synthetic polymer having pendant hydroxylgroups on the backbone of the polymer, the polymer having a molecularweight of at least about 1 kDa and less than or equal to about 1000 kDa,wherein the polymer is at least about 30% hydrolyzed and less than about95% hydrolyzed, or c) a polysorbate. The one or more surface alteringagents is present on the outer surface of the core particle at a densityof at least 0.01 molecules/nm². The one or more surface altering agentsis present in the pharmaceutical composition in an amount of betweenabout 0.001% to about 5% by weight. The plurality of coated particleshave an average smallest cross-sectional dimension of less than about 1micron. The pharmaceutical composition also includes one or moreophthalmically acceptable carriers, additives, and/or diluents.

In another set of embodiments, a method for treating inflammation,macular degeneration, macular edema, uveitis, dry eye, blepharitis,glaucoma, and/or other disorder in an eye of a patient is provided. Themethod involves administering to an eye of the patient, a pharmaceuticalcomposition comprising a plurality of coated particles, comprising acore particle comprising a pharmaceutical agent or a salt thereof,wherein the pharmaceutical agent or salt thereof constitutes at leastabout 80 wt % of the core particle, and wherein the pharmaceutical agentor salt thereof comprises bromfenac calcium, diclofenac free acid, orketorolac free acid. The plurality of coated particles also includes acoating comprising one or more surface-altering agents surrounding thecore particle, wherein the one or more surface-altering agents comprisesat least one of: a) a triblock copolymer comprising a hydrophilicblock-hydrophobic block-hydrophilic block configuration, wherein thehydrophobic block has a molecular weight of at least about 2 kDa, andthe hydrophilic blocks constitute at least about 15 wt % of the triblockcopolymer, b) a synthetic polymer having pendant hydroxyl groups on thebackbone of the polymer, the polymer having a molecular weight of atleast about 1 kDa and less than or equal to about 1000 kDa, wherein thepolymer is at least about 30% hydrolyzed and less than about 95%hydrolyzed, or c) a polysorbate. The one or more surface altering agentsis present on the outer surface of the core particle at a density of atleast 0.01 molecules/nm². The one or more surface altering agents ispresent in the pharmaceutical composition in an amount of between about0.001% to about 5% by weight. The plurality of coated particles have anaverage smallest cross-sectional dimension of less than about 1 micron.The pharmaceutical composition also includes one or more ophthalmicallyacceptable carriers, additives, and/or diluents.

In another set of embodiments, a pharmaceutical composition suitable foradministration to an eye is provided. The pharmaceutical compositioncomprises a plurality of coated particles, comprising a core particlecomprising a pharmaceutical agent or a salt thereof selected from thegroup consisting of a corticosteroid, a receptor tyrosine kinase (RTK)inhibitor, a cyclooxygenase (COX) inhibitor, an angiogenesis inhibitor,a prostaglandin analog, an NSAID, a beta blocker, and a carbonicanhydrase inhibitor. The plurality of coated particles also includes acoating comprising a surface-altering agent surrounding the coreparticle, wherein the one or more surface-altering agents comprises atleast one of: a) a triblock copolymer comprising a hydrophilicblock-hydrophobic block-hydrophilic block configuration, wherein thehydrophobic block has a molecular weight of at least about 2 kDa, andthe hydrophilic blocks constitute at least about 15 wt % of the triblockcopolymer, wherein the hydrophobic block associates with the surface ofthe core particle, and wherein the hydrophilic block is present at thesurface of the coated particle and renders the coated particlehydrophilic, b) a synthetic polymer having pendant hydroxyl groups onthe backbone of the polymer, the polymer having a molecular weight of atleast about 1 kDa and less than or equal to about 1000 kDa, wherein thepolymer is at least about 30% hydrolyzed and less than about 95%hydrolyzed, or c) a polysorbate. The one or more surface altering agentsis present on the outer surface of the core particle at a density of atleast 0.01 molecules/nm². The one or more surface altering agents ispresent in the pharmaceutical composition in an amount of between about0.001% to about 5% by weight. The pharmaceutical composition alsoincludes one or more ophthalmically acceptable carriers, additives,and/or diluents.

In another set of embodiments, a method of treating, diagnosing,preventing, or managing an ocular condition in a subject is provided.The method involves administering a composition to an eye of a subject,wherein the composition comprises a plurality of coated particles, thecoated particles comprising a core particle comprising a pharmaceuticalagent or a salt thereof selected from the group consisting of acorticosteroid, a receptor tyrosine kinase (RTK) inhibitor, acyclooxygenase (COX) inhibitor, an angiogenesis inhibitor, aprostaglandin analog, an NSAID, a beta blocker, and a carbonic anhydraseinhibitor, and a coating comprising one or more surface-altering agentssurrounding the core particle. The one or more surface-altering agentscomprises at least one of: a) a triblock copolymer comprising ahydrophilic block-hydrophobic block-hydrophilic block configuration,wherein the hydrophobic block has a molecular weight of at least about 2kDa, and the hydrophilic blocks constitute at least about 15 wt % of thetriblock copolymer, wherein the hydrophobic block associates with thesurface of the core particle, and wherein the hydrophilic block ispresent at the surface of the coated particle and renders the coatedparticle hydrophilic, or b) a synthetic polymer having pendant hydroxylgroups on the backbone of the polymer, the polymer having a molecularweight of at least about 1 kDa and less than or equal to about 1000 kDa,wherein the polymer is at least about 30% hydrolyzed and less than about95% hydrolyzed, or c) a polysorbate. The method involves delivering thepharmaceutical agent to a tissue in the eye of the subject.

In another set of embodiments, a method of improving the ocularbioavailability of a pharmaceutical agent in a subject is provided. Themethod involves administering a composition to an eye of the subject,wherein the composition comprises a plurality of coated particles. Thecoated particles comprise a core particle comprising a pharmaceuticalagent or a salt thereof selected from the group consisting of acorticosteroid, a receptor tyrosine kinase (RTK) inhibitor, acyclooxygenase (COX) inhibitor, an angiogenesis inhibitor, aprostaglandin analog, an NSAID, a beta blocker, and a carbonic anhydraseinhibitor, and a coating comprising a surface-altering agent surroundingthe core particle. The coating on the core particle is present in asufficient amount to improve the ocular bioavailability of thepharmaceutical agent when administered in the composition, compared tothe ocular bioavailability of the pharmaceutical agent when administeredas a core particle without the coating.

In another set of embodiments, a method of improving the concentrationof a pharmaceutical agent in a tissue of a subject is provided. Themethod involves administering a composition to an eye of the subject,wherein the composition comprises a plurality of coated particles. Thecoated particles comprise a core particle comprising the pharmaceuticalagent or a salt thereof, wherein the pharmaceutical agent is loteprednoletabonate, and a coating comprising a surface-altering agent surroundingthe core particle. The tissue is selected from the group consisting of aretina, a macula, a sclera, or a choroid. The coating on the coreparticle is present in a sufficient amount to increase the concentrationof the pharmaceutical agent by at least 10% in the tissue whenadministered in the composition, compared to the concentration of thepharmaceutical agent in the tissue when administered as a core particlewithout the coating.

In another set of embodiments, a method of treating an ocular conditionin a subject by repeated administration of a pharmaceutical compositionis provided. The method involves

administering two or more doses of a pharmaceutical compositioncomprising loteprednol etabonate to an eye of a subject, wherein theperiod between consecutive doses is at least about 4 hours, at leastabout 6 hours, at least about 8 hours, at least about 12 hours, at leastabout 36 hours, or at least about 48 hours, wherein the amount ofloteprednol etabonate delivered to a tissue of an eye is effective totreat an ocular condition in the subject.

In another set of embodiments, a method of treating an ocular conditionin a subject by repeated administration of a pharmaceutical compositionis provided. The method involves administering two or more doses of apharmaceutical composition comprising one or more pharmaceutical agentsto an eye of a subject, wherein the period between consecutive doses isat least about 4 hours, at least about 6 hours, at least about 8 hours,at least about 12 hours, at least about 36 hours, or at least about 48hours. The one or more pharmaceutical agents is selected from the groupconsisting of loteprednol etabonate, sorafenib, linifanib, MGCD-265,pazopanib, cediranib, axitinib, bromfenac calcium, diclofenac free acid,ketorolac free acid and a combination thereof. The amount of thepharmaceutical agent delivered to a tissue of an eye is effective totreat an ocular condition in the subject.

In another set of embodiments, a pharmaceutical composition suitable fortreating an anterior ocular disorder by administration to an eye isprovided. The pharmaceutical composition comprises a plurality of coatedparticles, comprising a core particle comprising a corticosteroid (e.g.,loteprednol etabonate), and a coating comprising one or moresurface-altering agents surrounding the core particle. The one or moresurface-altering agents comprises at least one of: a) a triblockcopolymer comprising a hydrophilic block-hydrophobic block-hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, b) a synthetic polymerhaving pendant hydroxyl groups on the backbone of the polymer, thepolymer having a molecular weight of at least about 1 kDa and less thanor equal to about 1000 kDa, wherein the polymer is at least about 30%hydrolyzed and less than about 95% hydrolyzed, orc) a polysorbate. Theplurality of coated particles have an average smallest cross-sectionaldimension of less than about 1 micron. The coating on the core particleis present in a sufficient amount to increase the concentration of thecorticosteroid (e.g., loteprednol etabonate) by at least 50% in ananterior component of the eye selected from the group consisting of acornea or aqueous humor 30 minutes after administration whenadministered to the eye, compared to the concentration of thecorticosteroid in the tissue when administered as a core particlewithout the coating.

In another set of embodiments, a method of treating an anterior oculardisorder by administration to an eye is provided. The method involvesadministering a composition to an eye of a subject, wherein thecomposition comprises a plurality of coated particles. The plurality ofcoated particles comprises a core particle comprising a corticosteroid(e.g., loteprednol etabonate), and a coating comprising one or moresurface-altering agents surrounding the core particle. The one or moresurface-altering agents comprises at least one of a) a triblockcopolymer comprising a hydrophilic block-hydrophobic block-hydrophilicblock configuration, wherein the hydrophobic block has a molecularweight of at least about 2 kDa, and the hydrophilic blocks constitute atleast about 15 wt % of the triblock copolymer, b) a synthetic polymerhaving pendant hydroxyl groups on the backbone of the polymer, thepolymer having a molecular weight of at least about 1 kDa and less thanor equal to about 1000 kDa, wherein the polymer is at least about 30%hydrolyzed and less than about 95% hydrolyzed, or c) a polysorbate Themethod involves sustaining an ophthalmically efficacious level of thecorticosteroid (e.g., loteprednol etabonate) in an anterior oculartissue selected from the group consisting of a palpebral conjunctiva, abulbar conjunctiva, or a cornea for at least 12 hours afteradministration.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a schematic drawing of a mucus-penetrating particle having acoating and a core according to one set of embodiments;

FIG. 2A is a plot showing the ensemble averaged velocity <V_(mean)> inhuman cervicovaginal mucus (CVM) for 200 nm carboxylated polystyreneparticles (negative control), 200 nm PEGylated polystyrene particles(positive control), and nanocrystal particles (sample) made by millingand coated with different surface-altering agents according to one setof embodiments;

FIG. 2B is a plot showing the relative velocity <V_(mean)>_(rel) in CVMfor nanocrystal particles made by milling and coated with differentsurface-altering agents according to one set of embodiments;

FIGS. 3A-3D are histograms showing distribution of trajectory-meanvelocity V_(mean) in CVM within an ensemble of nanocrystal particlescoated with different surface-altering agents according to one set ofembodiments;

FIG. 4 is a plot showing <V_(mean)>_(rel) in CVM for nanocrystalparticles coated with different PEO-PPO-PEO Pluronic® triblockcopolymers, mapped with respect to molecular weight of the PPO block andthe PEO weight content (%), according to one set of embodiments;

FIG. 5 is a plot showing the mass transport through CVM for solidparticles having different core materials that are coated with eitherPluronic® F127 (MPP, mucus-penetrating particles) or sodium dodecylsulfate (CP, conventional particles, a negative control), according toone set of embodiments;

FIGS. 6A-6C show drug levels of loteprednol etabonate in the palpebralconjunctiva (FIG. 6A), bulbar conjunctiva (FIG. 6B), and cornea (FIG.6C) of New Zealand white rabbits after administration of commercialprescription loteprednol etabonate, Lotemax®, or particles ofloteprednol etabonate that were coated with Pluronic® F127, according toone set of embodiments;

FIG. 7A is a plot showing the ensemble averaged velocity <V_(mean)> inhuman cervicovaginal mucus (CVM) for PSCOO⁻ particles coated withvarious poly(vinyl alcohols) (PVAs) according to one set of embodiments;

FIG. 7B is a plot showing the relative velocity <V_(mean)>_(rel) in CVMfor PSCOO⁻ particles coated with various PVAs according to one set ofembodiments;

FIG. 8 is a plot showing relative velocity <V_(mean)>_(rel) in CVM forPSCOO⁻ particles incubated with various PVAs mapped according to thePVA's molecular weight and degree of hydrolysis, according to one set ofembodiments. Each data point represents <V_(mean)>_(rel) for theparticles stabilized with a specific PVA.

FIGS. 9A-9B are plots showing bulk transport in CVM in vitro of PSCOOnanoparticles coated with various PVAs, according to one set ofembodiments. Negative controls are uncoated 200 nm PSCOO particles;Positive controls are 200 nm PSCOO particles coated with Pluronic® F127.FIGS. 9A-9B represent data obtained with two different CVM samples;

FIGS. 10A-10B are plots showing ensemble-average velocity <V_(mean)>(FIG. 10A) and relative sample velocity <V_(mean)>_(rel) (FIG. 10B) forpoly(lactic acid) (PLA) nanoparticles (sample) prepared byemulsification with various PVAs as measured by multiple-particletracking in CVM, according to one set of embodiments;

FIG. 11 is a plot showing relative velocity <V_(mean)>_(rel) in CVM forPLA nanoparticles prepared by emulsification with various PVA's mappedaccording to the PVA's molecular weight and degree of hydrolysis,according to one set of embodiments. Each data point represents<V_(mean)>_(rel) of the particles stabilized with a specific PVA;

FIGS. 12A-12B are plots showing ensemble-average velocity <V_(mean)>(FIG. 12A) and relative sample velocity <V_(mean)>_(rel) (FIG. 12B) forpyrene nanoparticles (sample) and controls as measured bymultiple-particle tracking in CVM, according to one set of embodiments;

FIGS. 13A-13F are representative CVM velocity (V_(mean)) distributionhistograms for pyrene/nanocrystals obtained with varioussurface-altering agents (SAMPLE=Pyrene nanoparticles, POSITIVE=200 nmPS-PEG5K, NEGATIVE=200 nm PS-COO); according to one set of embodiments;

FIG. 14 is a plot of relative velocity <V_(mean)>_(rel) for pyrenenanocrystals coated with PVA in CVM mapped according to the PVA'smolecular weight and degree of hydrolysis according to one set ofembodiments;

FIGS. 15A-15B are schematic drawings showing the constituent parts (FIG.15A), including the mucus layers (FIG. 15B), of an eye, according to oneset of embodiments;

FIG. 15C is a schematic drawing showing MPP and CP in the ocular mucuslayer after topical instillation, according to one set of embodiments.The MPP may readily penetrate the outer mucus layer toward theglycocalyx while CP may be immobilized in the outer layer of mucus. Theclearance of the outer layer by the body's natural clearance mechanismsmay be accompanied by CP removal whereas MPP are retained in the lessrapidly cleared glycocalyx, leading to prolonged residence at the ocularsurface.

FIG. 16 is a schematic drawing illustrating three main pathways throughwhich a topically applied drug may be transported to the back of theeye, according to one set of embodiments.

FIGS. 17A-17B are plots showing that the levels of loteprednol etabonate(LE) in the cornea after an administration of LE MPP formulations arehigher than the levels of LE after an administration of a commercialformulation in an ocular PK study, according to one set of embodiments.Equivalent drug doses were topically administered to the eyes of NZWrabbit at t=0;

FIG. 18 is a plot showing distribution of MPP (loteprednol etabonatenanocrystals coated with Pluronic® F127) in ocular tissues in vivo 30minutes after eye drop instillation, according to one set ofembodiments. Rabbits were given one 50 μL dose of 0.5% loteprednoletabonate MPP formulation or a commercial drop, Lotemax®, in each eye.Retina, choroid, and sclera are sampled where the human macula would belocated. Error bars show standard errors of the mean (SEM, n=6);

FIG. 19 is a bar graph showing the density of Pluronic® F127 on thesurface of fluticasone propionate and loteprednol etabonatenanocrystals, according to one set of embodiments;

FIG. 20 is a plot showing CVM mobility scores for loteprednol etabonatenanoparticles obtained by milling in the presence of differentPEO-PPO-PEO Pluronic® triblock copolymers, mapped with respect tomolecular weight of the PPO block and the PEO weight content (%),according to one set of embodiments. The scoring criterion is asfollows: 0-0.5 immobile; 0.51-1.5 slightly mobile; 1.51-2.5 moderatelymobile; and 2.51-3.0 very mobile. Samples that failed to produce stablenanosuspensions are demarked with * and considered immobile (mobilityscore <0.5);

FIG. 21 is a plot showing the mass-transport-into-mucus data of thefollowing formulations: mucus penetrating particles comprisingloteprednol etabonate and Pluronic® F127 (LE F127), particles comprisingLoteprednol Etabonate and sodium dodecyl sulfate (LE SDS), and marketedformulation Lotemax®. The ratio of loteprednol etabonate to Pluronic®F127 1:1 wt % while the ratio of loteprednol etabonate to SDS is 50:1 wt%. Untreated 200 nm carboxylated polystyrene spheres and 200 nmcarboxylated polystyrene spheres treated with Pluronic® F127 wereemployed as the negative and positive control, respectively;

FIG. 22 shows the chemical structures of certain degradants ofloteprednol etabonate;

FIGS. 23A-23B are plots showing the pharmacokinetics (PK) of LE inocular tissues in vivo. Error bars show standard errors of the mean(n=6). FIG. 23A: Rabbits were given one 50 μL dose of 0.5% LE MPP or LESDS in each eye. FIG. 23B: Rabbits were given one 50 μL dose of 0.5%Lotemax® or LE SDS in each eye. Data for Lotemax® were obtained from aprevious experiment performed using the same techniques at the samefacility;

FIG. 24 is a plot showing the pharmacokinetics of LE in ocular tissuesin vivo. Rabbits were given one 50 μL dose of 0.5% Lotemax®,Lotemax®+F127, or LE MPPs in each eye. Error bars show standard errorsof the mean (n=6). Data for Lotemax® were obtained from a previousexperiment performed using the same techniques at the same facility;

FIG. 25 is a plot showing the pharmacokinetics of LE in ocular tissuesin vivo. Rabbits were given one 50 μL dose of 0.5% Lotemax® or 0.4% LEMPPs in each eye. Error bars show standard errors of the mean (n=6).

FIGS. 26A-26R are plots showing pharmacokinetics of LE and its two mainmetabolites, PJ-91 and PJ-90, in ocular tissues (e.g., conjunctiva,cornea, aqueous humor, iris and ciliary body (ICB), and central retina)and plasma in vivo. Rabbits were given one 50 μL dose of 0.5% Lotemax®or 0.4% LE MPP in each eye. Error bars show standard errors of the mean(n=6).

FIG. 27 is a plot showing in vitro release profiles for variousPEGylated MPPs loaded with fluticasone. Release conditions: 37° C., PBSwith 0.5% Tween 80.

FIGS. 28A-28B show representative 15-second trajectories of conventionalnanoparticles (FIG. 28A) and MPPs described herein (FIG. 28B) in humancervicovaginal mucus. The MPPs avoided entrapment and were able todiffuse through mucus.

FIGS. 29A-29B are plots showing sorafenib levels in cornea (FIG. 29A)and retina (FIG. 29B) of New Zealand white rabbits following a singletopical instillation of MPPs of sorafenib (e.g., MPP1 and MPP2) and anon-MPP comparator (e.g., an aqueous suspension of sorafenib). Errorbars show standard errors of the mean (n=6).

FIG. 30 is a plot showing the pharmacokinetics (PK) of LE in aqueoushumor in vivo. The rabbits were given one 35 μL dose of 0.5% Lotemax®gel or 0.4% LE MPPs in each eye. Error bars show standard errors of themean (n=6).

FIG. 31A is a plot showing the pharmacokinetics of LE in the aqueoushumor of New Zealand white rabbits in vivo. The rabbits were given one50 μL dose of different percentages of LE MPPs in each eye. Error barsshow standard errors of the mean (n=6). FIG. 31B is a plot showing theAUC₀₋₆ of LE in the aqueous humor of the NZW rabbits in vivo. Therabbits were given one 50 μL dose of different percentages of LE MPPs ineach eye.

FIG. 32 is a plot showing the stability of LE MPPs in the presence ofionic components, such as sodium chloride. Triangles: 0.45% sodiumchloride. Squares: 0.9% sodium chloride. LE MPPs were monitored bydynamic light scattering (DLS). Sizes at Week −1 represent particlesizes of the LE MPPs immediately after milling and before the LE MPPswere diluted to the final concentration on Week 0. FIG. 32 indicatesthat LE MPPs in the presence of sodium chloride were stable.

FIG. 33A is a plot showing the particle stability of LE MPPs. Twosamples of LE MPPs were monitored by dynamic light scattering (DLS). Onesample had been exposed to 25 kGy of gamma irradiation, and the othersample had not been exposed to gamma irradiation. FIG. 33B is a plotshowing the pharmacokinetics of LE in the cornea of New Zealand whiterabbits in vivo. The rabbits were given one 50 μL dose of LE MPPsincluding 0.4% LE in each eye of the rabbit. Error bars show standarderrors of the mean (n=6).

FIG. 34 is a plot showing an exemplary particle size distribution ofbromfenac calcium MPPs in formulations containing the MPPs. Bromfenaccalcium was milled in water, 125 mM of CaCl₂, or 50 mM of Tris buffer.The particle sizes were measured by dynamic light scattering. All threeformulations had a Z-average diameter of about 200 nm and apolydispersity index <0.2.

FIGS. 35A-35D are plots showing that MPPs including bromfenac calciumare stable over extended period of time when stored at room temperature.FIG. 35A: particle Z-average size of bromfenac calcium (bromfenac-Ca)MPPs diluted to a concentration of 0.09% w/v bromfenac-Ca and 0.09% w/vPluronic®F127 (F127) and stored at room temperature for 23 days. FIG.35B: polydispersity index of bromfenac-Ca MPPs diluted to aconcentration of 0.09% w/v bromfenac-Ca and 0.09% w/v F127 and stored atroom temperature for 23 days. FIG. 35C: particle Z-average size ofbromfenac-Ca MPPs diluted to a concentration of 0.09% w/v bromfenac-Caand 0.5% w/v F127 and stored at room temperature for 7 or 12 days. FIG.35D: polydispersity index of bromfenac-Ca MPPs diluted to aconcentration of 0.09% w/v bromfenac-Ca and 0.5% w/v F127 and stored atroom temperature for 7 or 12 days.

FIGS. 36A-36B are plots showing the pharmacokinetics of sorafenib andlinifanib in center-punch retina of New Zealand white rabbits in vivo.FIG. 36A: the rabbits were given one 50 μL dose of 0.5% sorafenib-MPP or0.5% sorafenib non-MPP control in each eye. Error bars show standarderrors of the mean (n=6). FIG. 36B: the rabbits were given one 50 μLdose of 2% linifanib-MPP or 2% linifanib non-MPP control in each eye.Error bars show standard errors of the mean (n=6).

FIGS. 37A-37B are plots showing the pharmacokinetics of pazopanib andMGCD-265 in center-punch retina of New Zealand white rabbits in vivo.FIG. 37A: the rabbits were given one 50 μL dose of 0.5% pazopanib-MPP ineach eye. Error bars show standard errors of the mean (n=6). CellularIC₅₀ is also shown for reference. FIG. 37B: the rabbits were given one50 μL dose of 2% MGCD-265-MPP in each eye. Error bars show standarderrors of the mean (n=6). Cellular IC50 is also shown for reference.

FIGS. 38A-38B are plots showing the pharmacokinetics of cediranib inocular tissues of HY79b pigmented rabbits in vivo. FIG. 38A: the rabbitswere given one 50 μL dose of 2% cediranib-MPP in the choroid. Error barsshow standard errors of the mean (n=6). Cellular IC50 is also shown forreference. FIG. 38B: the rabbits were given one 50 μL dose of 2%cediranib-MPP in the retina. Error bars show standard errors of the mean(n=6). Cellular IC50 is also shown for reference.

FIGS. 39A-39B are plots showing the pharmacokinetics of axitinib inocular tissues of Dutch belted rabbits in vivo. FIG. 39A: the rabbitswere given one 50 μL dose of 2% axitinib-MPP in the choroid. Error barsshow standard errors of the mean (n=6). Cellular IC50 is also shown forreference. FIG. 39B: the rabbits were given one 50 μL dose of 2%axitinib-MPP in the retina. Error bars show standard errors of the mean(n=6). Cellular IC50 is also shown for reference.

FIGS. 40A-40C are images showing the efficacy of axitinib-MPP in arabbit VEGF (vascular endothelial growth factor receptor)-challengemodel. Dutch belted rabbits were dosed with 50 μL of 5% axitinib-MPPevery 4 hours on days 1-6. On day 3, the rabbits received anintravitreal injection of VEGF. On day 6, the rabbits were accessed forleakage via fluorescein angiography. Rabbits in the vehicle (negativecontrol) group received vehicle every 4 hours on days 1-6. Rabbits inthe Avastin® (positive control) group received one intravitrealinjection of Avastin® on day 1.

FIG. 41 is a bar graph showing the bulk transport of particlescontaining diclofenac into human cervicovaginal mucus. Polystyreneparticles containing no surface-altering agents (PS) were used as anegative, non-MPP control. Particles containing polystyrene in the coreand Pluronic® F127 as the surface-altering agents (PS F127) were used asa positive, MPP control. Diclofenac F127 stands for particles containingdiclofenac in the core and Pluronic® F127 as the surface-alteringagents. Diclofenac SDS stands for particles containing diclofenac in thecore and sodium dodecyl sulfate (SDS) as the surface-altering agents.

FIG. 42 is a plot showing the pharmacokinetics of Loteprednol Etabonate(LE) in the cornea of New Zealand white rabbits in vivo. Rabbits weregiven one 50 μL dose of each one of the three LE-MPPs (i.e., LE-F127,LE-Tween80, and LE-PVA) or Lotemax® in each eye. The PVA had a molecularweight of about 2 kDa and was about 75% hydrolyzed. The dose of LE was0.5% in all instances. Error bars show standard errors of the mean(n=6).

DETAILED DESCRIPTION

Particles, compositions, and methods that aid particle transport inmucus are provided. The particles, compositions, and methods may beused, in some instances, for ophthalmic and/or other applications. Insome embodiments, the compositions and methods may involve modifying thesurface coatings of particles, such as particles of pharmaceuticalagents that have a low aqueous solubility. Such compositions and methodscan be used to achieve efficient transport of particles ofpharmaceutical agents though mucus barriers in the body for a widespectrum of applications, including drug delivery, imaging, anddiagnostic applications. In certain embodiments, a pharmaceuticalcomposition including such particles is well-suited for ophthalmicapplications, and may be used for delivering pharmaceutical agents tothe front of the eye, middle of the eye, and/or the back of the eye.

Particles having efficient transport through mucus barriers may bereferred to herein as mucus-penetrating particles (MPPs). Such particlesmay include surfaces that are modified with one or more surface-alteringagents that reduce the adhesion of the particles to mucus, or otherwiseincrease the transport of the particles through mucus barriers comparedto conventional particles or non-MPPs, i.e., particles that do notinclude such surface-altering agent(s).

In some embodiments, the particles comprise a corticosteroid such asloteprednol etabonate for treating an eye disease or condition. Thecorticosteroid may be present, for example, in the core of the particle.The particles include a surface-altering agent that modifies the surfaceof the particles to reduce the adhesion of the particles to mucus and/orto facilitate penetration of the particles through physiological mucus.Compositions including such particles are also provided, includingcompositions that can be administered topically to the eye. Suchcompositions are advantageous over marketed formulations, such asLotemax® or Alrex®, as the compositions described herein are able tomore readily penetrate the mucus layer of an ocular tissue to avoid orminimize mucus adhesion and/or rapid mucus clearance. Therefore, thecompositions may be more effectively delivered to and may be retainedlonger in the target issue. As a result, the compositions describedherein may be administered at a lower dose and/or less frequently thanmarketed formulations to achieve similar or superior exposure. Moreover,the relatively low and/or infrequent dosage of the compositions mayresult in fewer or less severe side effects, a more desirable toxicityprofile, and/or improved patient compliance. Other advantages areprovided below.

In some embodiments, the particles comprise one or more receptortyrosine kinase inhibitors (RTKi), such as sorafenib, linifanib,MGCD-265, pazopanib, cediranib, axitinib, or a combination thereof, fortreating an eye disease or condition. The one or more RTKi may bepresent, for example, in the core of the particle. Compositionsincluding such particles are also provided, including compositions thatcan be administered topically to the eye. For reasons described herein,such compositions may have advantages over certain conventionalformulations (e.g., an aqueous suspension of the respective RTKi).

In some embodiments, the particles comprise a non-steroidalanti-inflammatory drug (NSAID), such as a divalent metal salt ofbromfenac (e.g., a divalent metal salt of bromfenac, such as bromfenaccalcium)), diclofenac (e.g., diclofenac free acid or a divalent ortrivalent metal salt thereof, such as an alkaline earth metal salt ofdiclofenac), or ketorolac (e.g., ketorolac free acid or a divalent ortrivalent metal salt thereof, such as an alkaline earth metal salt ofketorolac), for treating an eye disease or condition. The NSAID may bepresent, for example, in the core of the particle. Compositionsincluding such particles are also provided, including compositions thatcan be administered topically to the eye. For reasons described herein,such compositions may have advantages over certain conventionalformulations (e.g., an aqueous solution of bromfenac sodium).

As described in more detail below, in some embodiments, the particles,compositions and/or formulations described herein may be used todiagnose, prevent, treat or manage diseases or conditions at the back ofthe eye, such as at the retina, macula, choroid, sclera and/or uvea,and/or diseases and conditions at the front and/or middle of the eye,such as at the cornea, conjunctiva (including palpebral and bulbar),iris and ciliary body. In some embodiments, the particles, compositionsand/or formulations are designed to be administered topically to theeye. In other embodiments, the particles, compositions and/orformulations are designed to be administered by direct injection intothe eye.

Delivering drugs to the eye topically is challenging due to the limitedpermeability of the cornea and sclera (the tissues exposed to aninstillation) and the eye's natural clearance mechanisms: drugsolutions, such as in conventional ophthalmic solutions, are typicallywashed away from the surface of the eye very rapidly by drainage andlachrymation; and drug particles, such as in conventional ophthalmicsuspensions, are typically trapped by the rapidly cleared mucus layer ofthe eye, and hence, are also rapidly cleared. Therefore, conventionalophthalmic solutions and suspensions currently used to treat conditionsat the front of the eye are typically administered at high doses andhigh frequency in order to achieve and sustain efficacy. Such frequenthigh dosing greatly reduces patient compliance and increases the risk oflocal adverse effects. Topically delivering drugs to the back of the eyeis even more challenging due to the lack of direct exposure to a topicalinstillation and because of the anatomical and physiological barriersassociated with this part of the eye. Consequently, little (if any) drugreaches the back of the eye when administered as conventional topicalophthalmic solutions or suspensions. Therefore, invasive deliverytechniques, such as intravitreal or perocular injections, are currentlyused for conditions at the back of the eye.

In some instances, the particles, compositions and/or formulationsdescribed herein that are mucus-penetrating can address these issuesassociated with delivery to the front of the eye (e.g., dosagefrequency) and the back of the eye (e.g., sufficient delivery) since theparticles may avoid adhesion to the mucus layer and/or may be moreevenly spread across the surface of the eye, thereby avoiding the eye'snatural clearance mechanisms and prolonging their residence at theocular surface. In some embodiments, the particles may effectivelypenetrate through physiological mucus to facilitate sustained drugrelease directly to the underlying tissues, as described in more detailbelow.

In some embodiments, the particles described herein have a core-shelltype arrangement. The core may comprise any suitable material such as asolid pharmaceutical agent or a salt thereof having a relatively lowaqueous solubility, a polymeric carrier, a lipid, and/or a protein. Thecore may also comprise a gel or a liquid in some embodiments. The coremay be coated with a coating or shell comprising a surface-alteringagent that facilitates mobility of the particle in mucus. As describedin more detail below, in some embodiments the surface-altering agent maycomprise a polymer (e.g., a synthetic or a natural polymer) havingpendant hydroxyl groups on the backbone of the polymer. The molecularweight and/or degree of hydrolysis of the polymer may be chosen toimpart certain transport characteristics to the particles, such asincreased transport through mucus. In certain embodiments, thesurface-altering agent may comprise a triblock copolymer comprising ahydrophilic block-hydrophobic block-hydrophilic block configuration. Themolecular weights of each of the blocks may be chosen to impart certaintransport characteristics to the particles, such as increased transportthrough mucus.

Non-limiting examples of particles are now provided. As shown in theillustrative embodiment of FIG. 1, a particle 10 includes a core 16(which may be in the form of a particle, referred to herein as a coreparticle) and a coating 20 surrounding the core. In one set ofembodiments, a substantial portion of the core is formed of one or moresolid pharmaceutical agents (e.g., a drug, therapeutic agent, diagnosticagent, imaging agent) that can lead to certain beneficial and/ortherapeutic effects. The core may be, for example, a nanocrystal (i.e.,a nanocrystal particle) of a pharmaceutical agent. In other embodiments,the core may include a polymeric carrier, optionally with one or morepharmaceutical agents encapsulated or otherwise associated with thecore. In yet other cases, the core may include a lipid, a protein, agel, a liquid, and/or another suitable material to be delivered to asubject. The core includes a surface 24 to which one or moresurface-altering agents can be attached. For instance, in some cases,core 16 is surrounded by coating 20, which includes an inner surface 28and an outer surface 32. The coating may be formed, at least in part, ofone or more surface-altering agents 34, such as a polymer (e.g., a blockcopolymer and/or a polymer having pendant hydroxyl groups), which mayassociate with surface 24 of the core. Surface-altering agent 34 may beassociated with the core particle by, for example, being covalentlyattached to the core particle, non-covalently attached to the coreparticle, adsorbed to the core, or attached to the core through ionicinteractions, hydrophobic and/or hydrophilic interactions, electrostaticinteractions, van der Waals interactions, or combinations thereof. Inone set of embodiments, the surface-altering agents, or portionsthereof, are chosen to facilitate transport of the particle through amucosal barrier (e.g., mucus or a mucosal membrane). In certainembodiments described herein, one or more surface-altering agents 34 areoriented in a particular configuration in the coating of the particle.For example, in some embodiments in which a surface-altering agent is atriblock copolymer, such as a triblock copolymer having a hydrophilicblock-hydrophobic block-hydrophilic block configuration, a hydrophobicblock may be oriented towards the surface of the core, and hydrophilicblocks may be oriented away from the core surface (e.g., towards theexterior of the particle). The hydrophilic blocks may havecharacteristics that facilitate transport of the particle through amucosal barrier, as described in more detail below.

Particle 10 may optionally include one or more components 40 such astargeting moieties, proteins, nucleic acids, and bioactive agents whichmay optionally impart specificity to the particle. For example, atargeting agent or molecule (e.g., a protein, nucleic acid, nucleic acidanalog, carbohydrate, or small molecule), if present, may aid indirecting the particle to a specific location in the subject's body. Thelocation may be, for example, a tissue, a particular cell type, or asubcellular compartment. One or more components 40, if present, may beassociated with the core, the coating, or both; e.g., they may beassociated with surface 24 of the core, inner surface 28 of the coating,outer surface 32 of the coating, and/or embedded in the coating. The oneor more components 40 may be associated through covalent bonds,absorption, or attached through ionic interactions, hydrophobic and/orhydrophilic interactions, electrostatic interactions, van der Waalsinteractions, or combinations thereof. In some embodiments, a componentmay be attached (e.g., covalently) to one or more of thesurface-altering agents of the coated particle using methods known tothose of ordinary skill in the art.

It should be understood that components and configurations other thanthose shown in FIG. 1 or described herein may be suitable for certainparticles and compositions, and that not all of the components shown inFIG. 1 are necessarily present in some embodiments.

In one set of embodiments, particle 10, when introduced into a subject,may interact with one or more components in the subject such as mucus,cells, tissues, organs, particles, fluids (e.g., blood), portionsthereof, and combinations thereof. In some such embodiments, the coatingof particle 10 can be designed to include surface-altering agents orother components with properties that allow favorable interactions(e.g., transport, binding, adsorption) with one or more materials fromthe subject. For example, the coating may include surface-alteringagents or other components having a certain hydrophilicity,hydrophobicity, surface charge, functional group, specificity forbinding, and/or density to facilitate or reduce particular interactionsin the subject. One specific example includes choosing a certainhydrophilicity, hydrophobicity, surface charge, functional group,specificity for binding, and/or density of one or more surface-alteringagents to reduce the physical and/or chemical interactions between theparticle and mucus of the subject, so as to enhance the mobility of theparticle through mucus. Other examples are described in more detailbelow.

In some embodiments, once a particle is successfully transported acrossa mucosal barrier (e.g., mucus or a mucosal membrane) in a subject,further interactions between the particle in the subject may take place.Interactions may take place, in some instances, through the coatingand/or the core, and may involve, for example, the exchange of materials(e.g., pharmaceutical agents, therapeutic agents, proteins, peptides,polypeptides, nucleic acids, nutrients, e.g.) from the one or morecomponents of the subject to particle 10, and/or from particle 10 to theone or more components of the subject. For example, in some embodimentsin which the core is formed of or comprises a pharmaceutical agent, thebreakdown, release and/or transport of the pharmaceutical agent from theparticle can lead to certain beneficial and/or therapeutic effects inthe subject. As such, the particles described herein can be used for thediagnosis, prevention, treatment or management of certain diseases orbodily conditions.

Specific examples for the use of the particles described herein areprovided below in the context of being suitable for administration to amucosal barrier (e.g., mucus or a mucosal membrane) in a subject. Itshould be appreciated that while many of the embodiments herein aredescribed in this context, and in the context of providing a benefit fordiseases and conditions that involve transport of materials across amucosal barrier, the invention is not limited as such and the particles,compositions, kits, and methods described herein may be used to prevent,treat, or manage other diseases or bodily conditions.

Mucus is a sticky viscoelastic gel that protects against pathogens,toxins, and debris at various points of entry into the body, includingthe eyes, nose, lungs, gastrointestinal tract, and female reproductivetract. Many synthetic nanoparticles are strongly mucoadhesive and becomeeffectively trapped in the rapidly-cleared peripheral mucus layer,vastly limiting their distribution throughout the mucosal membrane aswell as penetration toward the underlying tissue. The residence time ofthese trapped particles is limited by the turnover rate of theperipheral mucus layer, which, depending on the organ, ranges fromseconds to several hours. To ensure effective delivery of particlesincluding pharmaceutical agents (e.g., therapeutic, diagnostic, and/orimaging agents) via mucus membranes, such particles must be able toreadily diffuse through the mucus barrier, avoiding mucus adhesion. Asdescribed in more detail below, delivery of particles in mucus of theeye has particular challenges.

It has been recently demonstrated that modifying surfaces of polymericnanoparticles with a mucus-penetrating coating can minimize adhesion tomucus and thus allow rapid particle penetration across mucus barriers.Despite these improvements, only a handful of surface coatings have beenshown to facilitate mucus penetration of particles. Accordingly,improvements in compositions and methods involving mucus-penetratingparticles for delivery of pharmaceutical agents would be beneficial.

In some embodiments, the compositions and methods described hereininvolve mucus-penetrating particles without any polymeric carriers, orwith minimal use of polymeric carriers. Polymer-based mucus-penetratingparticles may have one or more inherent limitations in some embodiments.In particular, in light of drug delivery applications, these limitationsmay include one or more of the following: A) Low drug encapsulationefficiency and low drug loading: Encapsulation of drugs into polymericparticles is often inefficient, as generally less than 10% of the totalamount of drug used gets encapsulated into particles duringmanufacturing. Additionally, drug loadings above 50% are rarelyachieved. B) Convenience of usage: Formulations based on drug-loadedpolymeric particles, in general, typically need to be stored as drypowder to avoid premature drug release and, thus, require eitherpoint-of-use re-constitution or a sophisticated dosing device. C)Biocompatibility: Accumulation of slowly degrading polymer carriersfollowing repeated dosing and their toxicity over the long term presenta major concern for polymeric drug carriers. D) Chemical and physicalstability: Polymer degradation may compromise stability of encapsulateddrugs. In many encapsulation processes, the drug undergoes a transitionfrom a solution phase to a solid phase, which is not well-controlled interms of physical form of the emerging solid phase (i.e., amorphous vs.crystalline vs. crystalline polymorphs). This is a concern for multipleaspects of formulation performance, including physical and chemicalstability and release kinetics. E) Manufacturing complexity:Manufacturing, especially scalability, of drug-loaded polymeric MPPs isa fairly complex process that may involve multiple steps and aconsiderable amount of toxic organic solvents.

In some embodiments described herein, the compositions and methods ofmaking particles, including certain compositions and methods for makingparticles that have increased transport through mucosal barriers,address one or more, or all, of the concerns described above.Specifically, in some embodiments, the compositions and methods do notinvolve encapsulation into polymeric carriers or involve minimal use ofpolymeric carriers. Advantageously, by avoiding or minimizing the needto encapsulate pharmaceutical agents (e.g., drugs, imaging or diagnosticagents) into polymeric carriers, certain limitations of polymeric MPPswith respect to drug loading, convenience of usage, biocompatibility,stability, and/or complexity of manufacturing, may be addressed. Themethods and compositions described herein may facilitate clinicaldevelopment of the mucus-penetrating particle technology.

It should be appreciated, however, that in other embodiments,pharmaceutical agents may be associated with polymer carriers viaencapsulation or other processes. Thus, the description provided hereinis not limited in this respect. For instance, despite theabove-mentioned drawbacks of certain mucus-penetrating particlesincluding a polymeric carrier, in certain embodiments such particles maybe preferred. For example, it may be preferable to use polymer carriersfor controlled release purposes and/or for encapsulating certainpharmaceutical agents that are difficult to formulate into particles. Assuch, in some embodiments described herein, particles that include apolymer carrier are described.

As described in more detail below, in some embodiments, the compositionsand methods involve the use of PVAs that aids particle transport inmucus. The compositions and methods may involve making mucus-penetratingparticles (MPPs) by, for example, an emulsification process in thepresence of specific PVAs. In certain embodiments, the compositions andmethods involve making MPPs from pre-fabricated particles bynon-covalent coating with specific PVAs. In other embodiments, thecompositions and methods involve making MPPs in the presence of specificPVAs without any polymeric carriers, or with minimal use of polymericcarriers. It should be appreciated, however, that in other embodiments,polymeric carriers can be used.

PVA is a water-soluble non-ionic synthetic polymer. Due to its surfaceactive properties, PVA is widely used in the food and drug industries asa stabilizing agent for emulsions and, in particular, to enableencapsulation of a wide variety of compounds by emulsificationtechniques. PVA has the “generally recognized as safe” or “GRAS” statuswith the Food and Drug Administration (FDA), and has been used inauricular, intramuscular, intraocular, intravitreal, iontophoretic,ophthalmic, oral, topical, and transdermal drug products and/or drugdelivery systems.

In certain previous studies, many have described PVA as a mucoadhesivepolymer, suggesting or reporting that incorporating PVA in the particleformulation process leads to particles that are strongly mucoadhesive.Surprisingly, and contrary to the established opinion that PVA is amucoadhesive polymer, the inventors have discovered within the contextof the invention that compositions and methods utilizing specific PVAgrades aid particle transport in mucus and are not mucoadhesive incertain applications described herein. Specifically, mucus-penetratingparticles can be prepared by tailoring the degree of hydrolysis and/ormolecular weight of the PVA, which was previously unknown. Thisdiscovery significantly broadens the arsenal of techniques andingredients applicable for manufacturing MPPs.

In some embodiments described herein, the compositions and methods ofmaking particles, including certain compositions and methods for makingparticles that have increased transport through mucosal barriers,address one or more, or all, of the concerns described above.

It should be appreciated that while some of the description herein mayrelate to the use of PVAs in coatings, in other embodiments, PVAs arenot used or are used in conjunction with other polymers. For example, insome embodiments, PEG, Pluronics®, and/or other surfactants (e.g., apolysorbate (e.g., Tween 80®)) may be included in the compositions andmethods described herein (in replace of or in addition to PVAs). Inother embodiments, other polymers, such as those described in moredetail herein, may be used in coatings described herein.

As described in more detail below, in some embodiments, the compositionsand methods involve the use of poloxamers that aid particle transport inmucus. Poloxamers are typically nonionic triblock copolymers comprisinga central hydrophobic block (e.g., a poly(propylene oxide) block)flanked by two hydrophilic blocks (e.g., poly(ethylene oxide) blocks).Poloxamers have the trade name Pluronic®, examples of which are providedbelow

As described in more detail below, in certain embodiments, thecompositions and methods involve the use of polysorbates that aidparticle transport in mucus. Polysorbates are typically derived fromPEGylated sorbitan (a derivative of sorbitol) esterified with fattyacids. Common brand names for polysorbates include Tween®, Alkest®,Canarcel®. Examples of polysorbates include polyoxyethylene sorbitanmonooleate (e.g., Tween 80®), polyoxyethylene sorbitan monostearate(e.g., Tween 60®), polyoxyethylene sorbitan monopalmitate (e.g., Tween40®), and polyoxyethylene sorbitan monolaurate (e.g., Tween 20®).

Core Particles

As described above in reference to FIG. 1, particle 10 may include acore 16. The core may be formed of any suitable material, such as anorganic material, an inorganic material, a polymer, a lipid, a proteinor combinations thereof. In one set of embodiments, the core comprises asolid. The solid may be, for example, a crystalline or an amorphoussolid, such as a crystalline or amorphous solid pharmaceutical agent(e.g., a therapeutic agent, diagnostic agent, and/or imaging agent), ora salt thereof. In other embodiments, the core may comprise a gel or aliquid (e.g., an oil-in-water or water-in-oil emulsion). In someembodiments, more than one pharmaceutical agents may be present in thecore. Specific examples of pharmaceutical agents are provided in moredetail below.

The pharmaceutical agent may be present in the core in any suitableamount, e.g., at least about 0.01 wt %, at least about 0.1 wt %, atleast about 1 wt %, at least about 5 wt %, at least about 10 wt %, atleast about 20 wt %, at least about 30 wt %, at least about 40 wt %, atleast about 50 wt %, at least about 60 wt %, at least about 70 wt %, atleast about 80 wt %, at least about 85 wt %, at least about 90 wt %, atleast about 95 wt %, or at least about 99 wt % of the core. In oneembodiment, the core is formed of 100 wt % of the pharmaceutical agent.In some cases, the pharmaceutical agent may be present in the core atless than or equal to about 100 wt %, less than or equal to about 90 wt%, less than or equal to about 80 wt %, less than or equal to about 70wt %, less than or equal to about 60 wt %, less than or equal to about50 wt %, less than or equal to about 40 wt %, less than or equal toabout 30 wt %, less than or equal to about 20 wt %, less than or equalto about 10 wt %, less than or equal to about 5 wt %, less than or equalto about 2 wt %, or less than or equal to about 1 wt %. Combinations ofthe above-referenced ranges are also possible (e.g., present in anamount of at least about 80 wt % and less than or equal to about 100 wt%). Other ranges are also possible.

In embodiments in which the core particles comprise relatively highamounts of a pharmaceutical agent (e.g., at least about 50 wt % of thecore particle), the core particles generally have an increased loadingof the pharmaceutical agent compared to particles that are formed byencapsulating agents into polymeric carriers. This is an advantage fordrug delivery applications, since higher drug loadings mean that fewernumbers of particles may be needed to achieve a desired effect comparedto the use of particles containing polymeric carriers.

As described herein, in other embodiments in which a relatively highamounts of a polymer or other material forms the core, less amounts ofpharmaceutical agent may be present in the core.

The core may be formed of solid materials having various aqueoussolubilities (i.e., a solubility in water, optionally with one or morebuffers), and/or various solubilities in the solution in which the solidmaterial is being coated with a surface-altering agent. For example, thesolid material may have an aqueous solubility (or a solubility in acoating solution) of less than or equal to about 5 mg/mL, less than orequal to about 2 mg/mL, less than or equal to about 1 mg/mL, less thanor equal to about 0.5 mg/mL, less than or equal to about 0.1 mg/mL, lessthan or equal to about 0.05 mg/mL, less than or equal to about 0.01mg/mL, less than or equal to about 1 μg/mL, less than or equal to about0.1 μg/mL, less than or equal to about 0.01 μg/mL, less than or equal toabout 1 ng/mL, less than or equal to about 0.1 ng/mL, or less than orequal to about 0.01 ng/mL at 25° C. In some embodiments, the solidmaterial may have an aqueous solubility (or a solubility in a coatingsolution) of at least about 1 μg/mL, at least about 10 μg/mL, at leastabout 0.1 ng/mL, at least about 1 ng/mL, at least about 10 ng/mL, atleast about 0.1 μg/mL, at least about 1 μg/mL, at least about 5 μg/mL,at least about 0.01 mg/mL, at least about 0.05 mg/mL, at least about 0.1mg/mL, at least about 0.5 mg/mL, at least about 1.0 mg/mL, at leastabout 2 mg/mL. Combinations of the above-noted ranges are possible(e.g., an aqueous solubility or a solubility in a coating solution of atleast about 10 μg/mL and less than or equal to about 1 mg/mL). Otherranges are also possible. The solid material may have these or otherranges of aqueous solubilities at any point throughout the pH range(e.g., from pH 1 to pH 14).

In some embodiments, the core may be formed of a material within one ofthe ranges of solubilities classified by the U.S. PharmacopeiaConvention: e.g., very soluble: >1,000 mg/mL; freely soluble: 100-1,000mg/mL; soluble: 33-100 mg/mL; sparingly soluble: 10-33 mg/mL; slightlysoluble: 1-10 mg/mL; very slightly soluble: 0.1-1 mg/mL; and practicallyinsoluble: <0.1 mg/mL.

Although a core may be hydrophobic or hydrophilic, in many embodimentsdescribed herein, the core is substantially hydrophobic. “Hydrophobic”and “hydrophilic” are given their ordinary meaning in the art and, aswill be understood by those skilled in the art, in many instancesherein, are relative terms. Relative hydrophobicities andhydrophilicities of materials can be determined by measuring the contactangle of a water droplet on a planar surface of the substance to bemeasured, e.g., using an instrument such as a contact angle goniometerand a packed powder of the core material.

In some embodiments, a material (e.g., a material forming a particlecore) has a contact angle of at least about 20 degrees, at least about30 degrees, at least about 40 degrees, at least about 50 degrees, atleast about 60 degrees, at least about 70 degrees, at least about 80degrees, at least about 90 degrees, at least about 100 degrees, at leastabout 110 degrees, at least about 120 degrees, or at least about 130degrees. In some embodiments, a material has a contact angle of lessthan or equal to about 160 degrees, less than or equal to about 150degrees, less than or equal to about 140 degrees, less than or equal toabout 130 degrees, less than or equal to about 120 degrees, less than orequal to about 110 degrees, less than or equal to about 100 degrees,less than or equal to about 90 degrees, less than or equal to about 80degrees, or less than or equal to about 70 degrees. Combinations of theabove-referenced ranges are also possible (e.g., a contact angle of atleast about 30 degrees and less than or equal to about 120 degrees).Other ranges are also possible.

Contact angle measurements can be made using a variety of techniques;here a static contact angle measurement between a pellet of the startingmaterial which will be used to form the core and a bead of water isreferenced. The material used to form the core was received as a finepowder or otherwise was ground into a fine powder using a mortar andpestle. In order to form a surface on which to make measurements, thepowder was packed using a 7 mm pellet die set from International CrystalLabs. The material was added to the die and pressure was applied by handto pack the powder into a pellet, no pellet press or high pressure wasused. The pellet was then suspended for testing so that the top andbottom of the pellet (defined as the surface water is added to and theopposite parallel surface respectively) were not in contact with anysurface. This was done by not fully removing the pellet from the collarof the die set. The pellet therefore touches the collar on the sides andmakes no contact on the top or bottom. For contact angle measurements,water was added to the surface of the pellet until a bead of water witha steady contact angle over 30 seconds was obtained. The water was addedinto the bead of water by submerging or contacting the tip of thepipette or syringe used for addition to the bead of water. Once a stablebead of water was obtained, an image was taken and the contact angle wasmeasured using standard practices.

In embodiments in which the core comprises an inorganic material (e.g.,for use as imaging agents), the inorganic material may include, forexample, a metal (e.g., Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and othertransition metals), a semiconductor (e.g., silicon, silicon compoundsand alloys, cadmium selenide, cadmium sulfide, indium arsenide, andindium phosphide), or an insulator (e.g., ceramics such as siliconoxide). The inorganic material may be present in the core in anysuitable amount, e.g., at least about 1 wt %, at least about 5 wt %, atleast about 10 wt %, at least about 20 wt %, at least about 30 wt %, atleast about 40 wt %, at least about 50 wt %, at least about 75 wt %, atleast about 90 wt %, or at least about 99 wt %. In one embodiment, thecore is formed of 100 wt % inorganic material. In some cases, theinorganic material may be present in the core at less than or equal toabout 100 wt %, less than or equal to about 90 wt %, less than or equalto about 80 wt %, less than or equal to about 70 wt %, less than orequal to about 60 wt %, less than or equal to about 50 wt %, less thanor equal to about 40 wt %, less than or equal to about 30 wt %, lessthan or equal to about 20 wt %, less than or equal to about 10 wt %,less than or equal to about 5 wt %, less than or equal to about 2 wt %,or less than or equal to about 1 wt %. Combinations of theabove-referenced ranges are also possible (e.g., present in an amount ofat least about 1 wt % and less than or equal to about 20 wt %). Otherranges are also possible.

The core may, in some cases, be in the form of a quantum dot, a carbonnanotube, a carbon nanowire, or a carbon nanorod. In some cases, thecore comprises, or is formed of, a material that is not of biologicalorigin.

In some embodiments, the core includes one or more organic materialssuch as a synthetic polymer and/or a natural polymer. Examples ofsynthetic polymers include non-degradable polymers such aspolymethacrylate and degradable polymers such as polylactic acid,polyglycolic acid and copolymers thereof. Examples of natural polymersinclude hyaluronic acid, chitosan, and collagen. Other examples ofpolymers that may be suitable for portions of the core include thoseherein suitable for forming coatings on particles, as described below.In some cases, the one or more polymers present in the core may be usedto encapsulate or adsorb one or more pharmaceutical agents.

In certain embodiments, a core may include a pharmaceutical agentcomprising a lipid and/or a protein. Other materials are also possible.

If a polymer is present in the core, the polymer may be present in thecore in any suitable amount, e.g., less than or equal to about 100 wt %,less than or equal to about 90 wt %, less than or equal to about 80 wt%, less than or equal to about 70 wt %, less than or equal to about 60wt %, less than or equal to about 50 wt %, less than or equal to about40 wt %, less than or equal to about 30 wt %, less than or equal toabout 20 wt %, less than or equal to about 10 wt %, less than or equalto about 5 wt %, less than or equal to about 2 wt %, or less than orequal to about 1 wt %. In some cases, the polymer may be present in anamount of at least about 1 wt %, at least about 5 wt %, at least about10 wt %, at least about 20 wt %, at least about 30 wt %, at least about40 wt %, at least about 50 wt %, at least about 75 wt %, at least about90 wt %, or at least about 99 wt % in the core. Combinations of theabove-referenced ranges are also possible (e.g., present in an amount ofat least about 1 wt % and less than or equal to about 20 wt %). Otherranges are also possible. In one set of embodiments, the core is formedis substantially free of a polymeric component.

The core of a particle described herein may include a mixture of morethan one polymer. In some embodiments, the core, or at least a portionof the core, includes a mixture of a first polymer and a second polymer.In certain embodiments, the first polymer is a polymer described herein.In certain embodiments, the first polymer is a relatively hydrophobicpolymer (e.g., a polymer having a higher hydrophobicity than the secondpolymer). In certain embodiments, the first polymer is not a polyalkylether. In certain embodiments, the first polymer is polylactide (PLA),e.g., 100DL7A MW108K. In certain embodiments, the first polymer ispolylactide-co-glycolide (PLGA), e.g., PLGA1A MW4K. In otherembodiments, however, the first polymer may be a relatively hydrophilicpolymer (e.g., a polymer having a higher hydrophilicity than the secondpolymer).

In certain embodiments, the second polymer is a block copolymerdescribed herein (e.g., a diblock copolymer or a triblock copolymer). Incertain embodiments, the second polymer is a diblock copolymer includinga relatively hydrophilic block (e.g., a polyalkyl ether block) and arelatively hydrophobic block (e.g., a non-(polyalkyl ether) block). Incertain embodiments, the polyalkyl ether block of the second polymer isPEG (e.g., PEG2K or PEG5K). In certain embodiments, the non-(polyalkylether) block of the second polymer is PLA (e.g., 100DL9K, 100DL30, or100DL95). In certain embodiments, the non-(polyalkyl ether) block of thesecond polymer is PLGA (e.g., 8515PLGA54K, 7525PLGA15K, or 5050PLGA18K),In certain embodiments, the second polymer is 100DL9K-co-PEG2K. Incertain embodiments, the second polymer is 8515PLGA54K-co-PEG2K.

It should be appreciated that while “first” and “second” polymers aredescribed, in some embodiments, a particle or core described herein mayinclude only one such polymer. Additionally, while specific examples offirst and second polymers are provided, it should be appreciated thatother polymers, such as the polymers listed herein, can be used as firstor second polymers.

The first polymer and the relatively hydrophobic block of the secondpolymer may be the same or different polymer. In some cases, therelatively hydrophilic block of the second polymer is present primarilyat or on the surface of the core that includes the first and secondpolymers. For instance, the relatively hydrophilic block of the secondpolymer may act as a surface-altering agent as described herein. In somecases, the relatively hydrophobic block of the second polymer and thefirst polymer are present primarily inside the surface of the core thatincludes the first and second polymers. Additional details are providedin Example 19.

The relatively hydrophilic block (e.g., a polyalkyl ether block, such asPEG block) of the second polymer may have any suitable molecular weight.In certain embodiments, the molecular weight of the relativelyhydrophilic block of the second polymer is at least about 0.1 kDa, atleast about 0.2 kDa, at least about 0.5 kDa, at least about 1 kDa, atleast about 1.5 kDa, at least about 2 kDa, at least about 2.5 kDa, atleast about 3 kDa, at least about 4 kDa, at least about 5 kDa, at leastabout 6 kDa, at least about 8 kDa, at least about 10 kDa, at least about20 kDa, at least about 50 kDa, at least about 100 kDa, or at least about300 kDa. In certain embodiments, the molecular weight of the relativelyhydrophilic block of the second polymer is less than or equal to about300 kDa, less than or equal to about 100 kDa, less than or equal toabout 50 kDa, less than or equal to about 20 kDa, less than or equal toabout 10 kDa, less than or equal to about 8 kDa, less than or equal toabout 6 kDa, at least about 5 kDa, less than or equal to about 4 kDa,less than or equal to about 3 kDa, less than or equal to about 2.5 kDa,less than or equal to about 2 kDa, less than or equal to about 1.5 kDa,less than or equal to about 1 kDa, less than or equal to about 0.5 kDa,less than or equal to about 0.2 kDa, or less than or equal to about 0.1kDa. Combinations of the above-mentioned ranges are also possible (e.g.,at least about 0.5 kDa and less than or equal to about 10 kDa). Otherranges are also possible. In certain embodiments, the molecular weightof the relatively hydrophilic block of the second polymer is about 2kDa. In certain embodiments, the molecular weight of the relativelyhydrophilic block of the second polymer is about 5 kDa.

The relatively hydrophobic block (e.g., a non-(polyalkyl ether) block,such as PLGA or PLA block) of the second polymer may have any suitablemolecular weight. In certain embodiments, the relatively hydrophobicblock of the second polymer is relatively short in length and/or low inmolecular weight. In certain embodiments, the molecular weight of therelatively hydrophobic block of the second polymer is less than or equalto about 300 kDa, less than or equal to about 100 kDa, less than orequal to about 80 kDa, less than or equal to about 60 kDa, less than orequal to about 54 kDa, less than or equal to about 50 kDa, less than orequal to about 40 kDa, less than or equal to about 30 kDa, less than orequal to about 20 kDa, less than or equal to about 15 kDa less than orequal to about 10 kDa, less than or equal to about 5 kDa, less than orequal to about 2 kDa, or less than or equal to about 1 kDa. In certainembodiments, the molecular weight of the PLGA or PLA block of the secondpolymer is at least about 0.1 kDa, at least about 0.3 kDa, at leastabout 1 kDa, at least about 2 kDa, at least about 4 kDa, at least about6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa,at least about 10 kDa, at least about 12 kDa, at least about 15 kDa, atleast about 20 kDa, at least about 30 kDa, at least about 50 kDa, or atleast about 100 kDa. Combinations of the above-mentioned ranges are alsopossible (e.g., less than or equal to about 20 kDa and at least about 1kDa). Other ranges are also possible. In certain embodiments, themolecular weight of the relatively hydrophobic block of the secondpolymer is about 9 kDa.

The relatively hydrophilic block (e.g., a polyalkyl ether block, such asPEG block) of the second polymer may be present in any suitable amountor density at or on the surface of a core described herein. In certainembodiments, the PEG block of the second polymer is present at or on thesurface of the core at at least about 0.001, at least about 0.003, atleast about 0.03, at least about 0.1 at least about 0.15, at least about0.18, at least about 0.2, at least about 0.3, at least about 0.5, atleast about 1, at least about 3, at least about 30, or at least about100 PEG chains per nm² of the surface area of the core. In certainembodiments, the PEG block of the second polymer is present at or on thesurface of the core at less than or equal to about 100, less than orequal to about 30, less than or equal to about 10, less than or equal toabout 3, less than or equal to about 1, less than or equal to about 0.5,less than or equal to about 0.3, less than or equal to about 0.2, lessthan or equal to about 0.18, less than or equal to about 0.15, less thanor equal to about 0.1, less than or equal to about 0.03, less than orequal to about 0.01, less than or equal to about 0.003, or less than orequal to about 0.001 PEG chains per nm² of the surface area of the core.Combinations of the above-mentioned ranges are also possible (e.g., atleast about 0.03 and less than or equal to about 1 PEG chains per nm² ofthe surface area of the core). Other ranges are also possible. Incertain embodiments, the PEG block of the second polymer is present ator on the surface of the core at at least about 0.18 PEG chains per nm²of the surface area of the core.

The relatively hydrophilic block (e.g., a polyalkyl ether block, such asPEG block) of the second polymer may be present in any suitable amountin a particle or core described herein. In certain embodiments, therelatively hydrophilic block of the second polymer is present in thecore at less than or equal to about less than or equal to about 90 wt %,less than or equal to about 80 wt %, less than or equal to about 70 wt%, less than or equal to about 60 wt %, less than or equal to about 50wt %, less than or equal to about 40 wt %, less than or equal to about30 wt %, less than or equal to about 20 wt %, less than or equal toabout 10 wt %, less than or equal to about 5 wt %, less than or equal toabout 4 wt %, less than or equal to about 3 wt %, less than or equal toabout 2 wt %, less than or equal to about 1 wt %, less than or equal toabout 0.5 wt %, less than or equal to about 0.2 wt %, less than or equalto about 0.1 wt %, less than or equal to about 0.05 wt %, less than orequal to about 0.02 wt %, or less than or equal to about 0.01 wt % ofthe particle or core. In certain embodiments, the relatively hydrophilicblock of the second polymer is present in the core at at least about0.01 wt %, at least about 0.02 wt %, at least about 0.05 wt %, at leastabout 0.1 wt %, at least about 0.2 wt %, at least about 0.5 wt %, atleast about 1 wt %, at least about 2 wt %, at least about 3 wt %, atleast about 4 wt %, at least about 5 wt %, at least about 10 wt %, atleast about 20 wt %, at least about 30 wt %, at least about 40 wt %, atleast about 50 wt %, at least about 60 wt %, at least about 70 wt %, atleast about 80 wt %, or at least about 90 wt % of the particle or core.Combinations of the above-mentioned ranges are also possible (e.g., lessthan or equal to about 10 wt % and at least about 0.5 wt % of theparticle or core). Other ranges are also possible. In certainembodiments, the relatively hydrophilic block of the second polymer ispresent at less than or equal to about 3 wt % of the particle or core.

The relatively hydrophilic block (e.g., a polyalkyl ether block, such asPEG block) and the relatively hydrophobic block (e.g., a non-(polyalkylether) block, such as PLGA or PLA block) of the second polymer may bepresent in the core in any suitable ratio. In certain embodiments, theratio of the relatively hydrophilic block to relatively hydrophobicblock of the second polymers is at least about 1:99, at least about10:90, at least about 20:80, at least about 30:70, at least about 40:60,at least about 50:50, at least about 60:40, at least about 70:30, atleast about 80:20, at least about 90:10, or at least about 99:1 w/w. Incertain embodiments, the ratio of the relatively hydrophilic block torelatively hydrophobic block is less than or equal to about 99:1, lessthan or equal to about 90:10, less than or equal to about 80:20, lessthan or equal to about 70:30, less than or equal to about 60:40, lessthan or equal to about 50:50, less than or equal to about 40:60, lessthan or equal to about 30:70, less than or equal to about 20:80, lessthan or equal to about 10:90, or less than or equal to about 1:99 w/w.Combinations of the above-mentioned ranges are also possible (e.g.,greater than about 70:30 and less than or equal to about 90:10 w/w).Other ranges are also possible. In certain embodiments, the ratio of therelatively hydrophilic block to relatively hydrophobic block is about20:80 w/w.

The first polymer (e.g., PLA or PLGA) and the second polymer (e.g.,PLA-co-PEG or PLGA-co-PEG) may be present in the particle or core in anysuitable ratio. In certain embodiments, the ratio of the first polymerto second polymer in the particle or core is at least about 1:99, atleast about 10:90, at least about 20:80, at least about 30:70, at leastabout 40:60, at least about 50:50, at least about 60:40, at least about65:35, at least about 70:30, at least about 75:25, at least about 80:20,at least about 85:15, at least about 90:10, at least about 95:5, or atleast about 99:1 w/w. In certain embodiments, the ratio of the firstpolymer to second polymer in the particle or core is less than or equalto about 99:1, less than or equal to about 95:5, less than or equal toabout 90:10, less than or equal to about 85:15, less than or equal toabout 80:20, less than or equal to about 75:25, less than or equal toabout 70:30, less than or equal to about 65:35, less than or equal toabout 60:40, less than or equal to about 50:50, less than or equal toabout 40:60, less than or equal to about 30:70, less than or equal toabout 20:80, less than or equal to about 10:90, or less than or equal toabout 1:99 w/w. Combinations of the above-mentioned ranges are alsopossible (e.g., greater than about 70:30 and less than or equal to about90:10 w/w). Other ranges are also possible. In certain embodiments, theratio of the first polymer to second polymer in the particle or core isabout 70:30 w/w. In certain embodiments, the ratio of the first polymerto second polymer in the particle or core is about 80:20 w/w.

The particle or core comprising a mixture of the first polymer and thesecond polymer described herein may further include a coating describedherein. The coating may be at or on the surface of the particle (e.g.,the surface of the first polymer and/or the second polymer). In someembodiments, the coating includes a hydrophilic material. The coatingmay include one or more surface-altering agents described herein, suchas a polymer and/or a surfactant (e.g., a PVA, a poloxamer, apolysorbate (e.g., Tween 80®)).

The core may have any suitable shape and/or size. For instance, the coremay be substantially spherical, non-spherical, oval, rod-shaped,pyramidal, cube-like, disk-shaped, wire-like, or irregularly shaped. Thecore may have a largest or smallest cross-sectional dimension of, forexample, less than or equal to about 10 μm, less than or equal to about5 μm, less than or equal to about 1 μm, less than or equal to about 800nm, less than or equal to about 700 nm, less than or equal to about 500nm, less than or equal to 400 nm, less than or equal to 300 nm, lessthan or equal to about 200 nm, less than or equal to about 100 nm, lessthan or equal to about 75 nm, less than or equal to about 50 nm, lessthan or equal to about 40 nm, less than or equal to about 35 nm, lessthan or equal to about 30 nm, less than or equal to about 25 nm, lessthan or equal to about 20 nm, less than or equal to about 15 nm, or lessthan or equal to about 5 nm. In some cases, the core may have a largestor smallest cross-sectional dimension of, for example, at least about 5nm, at least about 20 nm, at least about 50 nm, at least about 100 nm,at least about 200 nm, at least about 300 nm, at least about 400 nm, atleast about 500 nm, at least about 1 μm, or at least about 5 μm.Combinations of the above-referenced ranges are also possible (e.g., alargest or smallest cross-sectional dimension of at least about 50 nmand less than or equal to about 500 nm). Other ranges are also possible.In some embodiments, the sizes of the cores formed by a processdescribed herein have a Gaussian-type distribution. Unless indicatedotherwise, the measurements of particle/core sizes herein refer to thesmallest cross-sectional dimension.

Those of ordinary skill in the art are familiar with techniques todetermine sizes (e.g., smallest or largest cross-sectional dimensions)of particles. Examples of suitable techniques include (DLS),transmission electron microscopy, scanning electron microscopy,electroresistance counting and laser diffraction. Other suitabletechniques are known to those or ordinary skill in the art. Althoughmany methods for determining sizes of particles are known, the sizesdescribed herein (e.g., average particle sizes, thicknesses) refer toones measured by dynamic light scattering.

Methods of Forming Core Particles and Coated Particles

The core particles described herein may be formed by any suitablemethod. Suitable methods may include, for example, so called top-downtechniques, i.e. techniques based on size reduction of relatively largeparticles into smaller particles (e.g., milling or homogenization) or socalled bottom-up techniques, i.e. techniques based on the growth ofparticles from smaller particles or individual molecules (e.g.,precipitation or spray-freezing into liquid).

In some embodiments, core particles may be coated with a coating. Forexample, core particles may be provided or formed in a first step, andthen the particles may be coated in a second step to form coatedparticles. In other embodiments, core particles may be formed and coatedsubstantially simultaneously (e.g., in a single step). Examples of theseand other methods are provided below.

In some embodiments, the coated particles described herein are formed bya method that involves using a formulation process, a milling process,and/or a dilution process. In certain embodiments, a method of formingthe particles includes a milling process, optionally with a formulationprocess and/or a dilution process. A formulation process may be used toform a suspension or solution comprising a core material, one or moresurface-altering agents, and other components, such as solvents,tonicity agents, chelating agents, salts, preservatives, anti-microbialagents and/or buffers (e.g., a sodium citrate and citric acid buffer),each of which is as described herein. The formulation process may beperformed using a formulation vessel. The core material and othercomponents may be added into the formulation vessel at the same time ordifferent times. A mixture of the core material and/or one or more othercomponents may be stirred and/or shaken, or otherwise agitated in thevessel to facilitate suspending and/or dissolving the components. Thetemperature and/or pressure of the fluids containing the core material,the other components, and/or the mixture may also be individuallyincreased or decreased to facilitate the suspending and/or dissolvingprocesses. In some embodiments, the core material and other componentsare processed as described herein in the formulation vessel under aninert atmosphere (e.g., nitrogen or argon) and/or protected from light.The suspension or solution obtained from the formulation vessel may besubsequently subject to a milling process which may be followed by adilution process.

In some embodiments involving a core comprising a solid material, amilling process may be used to reduce the size of the solid material toform particles in the micrometer to nanometer size range. The millingprocess may be performed using a mill or other suitable apparatus. Dryand wet milling processes such as jet milling, cryo-milling, ballmilling, media milling, sonication, and homogenization are known and canbe used in methods described herein. Generally, in a wet millingprocess, a suspension of the material to be used as the core is agitatedwith or without excipients to reduce particle size. Dry milling is aprocess wherein the material to be used as the core is mixed withmilling media with or without excipients to reduce particle size. In acyro-milling process, a suspension of the material to be used as thecore is mixed with milling media with or without excipients under cooledtemperatures.

After milling or other suitable process for reducing the size of a corematerial, a dilution process may be used to form and/or modify coatedparticles from a suspension. The coated particles may comprise a corematerial, one or more surface-altering agents, and other components,such as solvents, tonicity agents, chelating agents, salts,preservatives, anti-microbial agents, and buffers (e.g., a sodiumcitrate and citric acid buffer). A dilution process may be used toachieve a target dosing concentration by diluting a solution orsuspension of particles that were coated during a milling step, with orwithout the additional of surface-altering agents and/or othercomponents. In certain embodiments, a dilution process may be used toexchange a first surface-altering agent with a second surface-alteringagent from a surface of a particle as described herein.

The dilution process may be performed using a product vessel or anyother suitable apparatus. In certain embodiments, the suspension isdiluted, i.e., mixed or otherwise processed with a diluent, in theproduct vessel. The diluent may contain solvents, surface-alteringagents, tonicity agents, chelating agents, salts, preservatives, oranti-microbial agents, or a combination thereof, as described herein.The suspension and the diluent may be added into the product vessel atthe same time or different times. In certain embodiments when thesuspension is obtained from a milling process involving milling media,the milling media may be separated from the suspension before thesuspension is added into the product vessel. The suspension, thediluent, or the mixture of the suspension and the diluent may be stirredand/or shaken, or otherwise agitated, to form the coated particlesdescribed herein. The temperature and/or pressure of the suspension, thediluent, or the mixture may also be individually increased or decreasedto form the coated particles. In some embodiments, the suspension andthe diluent are processed in the product vessel under an inertatmosphere (e.g., nitrogen or argon) and/or protected from light.

In some embodiments, the core particles described herein may be producedby milling of a solid material (e.g., a pharmaceutical agent) in thepresence of one or more surface-altering agents. Small particles of asolid material may require the presence of one or more surface-alteringagents, which may function as a stabilizer in some embodiments, in orderto stabilize a suspension of particles without agglomeration oraggregation in a liquid solution. In some such embodiments, thestabilizer may act as a surface-altering agent, forming a coating on theparticle.

As described herein, in some embodiments, a method of forming a coreparticle involves choosing a surface-altering agent that is suitable forboth milling and for forming a coating on the particle and rendering theparticle mucus penetrating. For example, as described in more detailbelow, it has been demonstrated that 200-500 nm nanoparticles of a modelcompound pyrene produced by milling of pyrene in the presence of certainPluronics® polymers resulted in particles that can penetratephysiological mucus samples at the same rate as well-establishedPEGylated polymeric MPPs. Interestingly, it was observed that only asubset of Pluronics® polymers tested fit the criteria of being suitablefor both milling and for forming a coating on the particle that rendersthe particle mucus penetrating, as described in more detail below.

In a wet milling process, milling can be performed in a dispersion(e.g., an aqueous dispersion) containing one or more surface-alteringagents, a grinding medium, a solid to be milled (e.g., a solidpharmaceutical agent), and a solvent. Any suitable amount of asurface-altering agent can be included in the solvent. In someembodiments, a surface-altering agent may be present in the solvent inan amount of at least about 0.001% (wt % or % weight to volume (w:v)),at least about 0.01%, at least about 0.1%, at least about 0.5%, at leastabout 1%, at least about 2%, at least about 3%, at least about 4%, atleast about 5%, at least about 6%, at least about 7%, at least about 8%,at least about 10%, at least about 12%, at least about 15%, at leastabout 20%, at least about 40%, at least about 60%, or at least about 80%of the solvent. In some cases, the surface-altering agent may be presentin the solvent in an amount of about 100% (e.g., in an instance wherethe surface-altering agent is the solvent). In other embodiments, thesurface-altering agent may be present in the solvent in an amount ofless than or equal to about 100%, less than or equal to about 80%, lessthan or equal to about 60%, less than or equal to about 40%, less thanor equal to about 20%, less than or equal to about 15%, less than orequal to about 12%, less than or equal to about 10%, less than or equalto about 8%, less than or equal to about 7%, less than or equal to about6%, less than or equal to about 5%, less than or equal to about 4%, lessthan or equal to about 3%, less than or equal to about 2%, or less thanor equal to about 1% of the solvent. Combinations of theabove-referenced ranges are also possible (e.g., an amount of less thanor equal to about 5% and at least about 1% of the solvent). Other rangesare also possible. In certain embodiments, the surface-altering agent ispresent in the solvent in an amount of about 0.01-2% of the solvent. Incertain embodiments, the surface-altering agent is present in thesolvent in an amount of about 0.2-20% of the solvent. In certainembodiments, the surface-altering agent is present in the solvent in anamount of about 0.1% of the solvent. In certain embodiments, thesurface-altering agent is present in the solvent in an amount of about0.4% of the solvent. In certain embodiments, the surface-altering agentis present in the solvent in an amount of about 1% of the solvent. Incertain embodiments, the surface-altering agent is present in thesolvent in an amount of about 2% of the solvent. In certain embodiments,the surface-altering agent is present in the solvent in an amount ofabout 5% of the solvent. In certain embodiments, the surface-alteringagent is present in the solvent in an amount of about 10% of thesolvent.

The particular range chosen may influence factors that may affect theability of the particles to penetrate mucus such as the stability of thecoating of the surface-altering agent on the particle surface, theaverage thickness of the coating of the surface-altering agent on theparticles, the orientation of the surface-altering agent on theparticles, the density of the surface altering agent on the particles,surface-altering agent:drug ratio, drug concentration, the size,dispersibility, and polydispersity of the particles formed, and themorphology of the particles formed.

The pharmaceutical agent (or salt thereof) may be present in the solventin any suitable amount. In some embodiments, the pharmaceutical agent(or salt thereof) is present in an amount of at least about 0.001% (wt %or % weight to volume (w:v)), at least about 0.01%, at least about 0.1%,at least about 0.5%, at least about 1%, at least about 2%, at leastabout 3%, at least about 4%, at least about 5%, at least about 6%, atleast about 7%, at least about 8%, at least about 10%, at least about12%, at least about 15%, at least about 20%, at least about 30%, atleast about 40%, at least about 60%, or at least about 80% of thesolvent. In some cases, the pharmaceutical agent (or salt thereof) maybe present in the solvent in an amount of less than or equal to about100%, less than or equal to about 90%, less than or equal to about 80%,less than or equal to about 60%, less than or equal to about 40%, lessthan or equal to about 30%, less than or equal to about 20%, less thanor equal to about 15%, less than or equal to about 12%, less than orequal to about 10%, less than or equal to about 8%, less than or equalto about 7%, less than or equal to about 6%, less than or equal to about5%, less than or equal to about 4%, less than or equal to about 3%, lessthan or equal to about 2%, or less than or equal to about 1% of thesolvent. Combinations of the above-referenced ranges are also possible(e.g., an amount of less than or equal to about 20% and at least about1% of the solvent). In some embodiments, the pharmaceutical agent ispresent in the above ranges but in w:v

The ratio of surface-altering agent to pharmaceutical agent (or saltthereof) in a solvent may also vary. In some embodiments, the ratio ofsurface-altering agent to pharmaceutical agent (or salt thereof) may beat least 0.001:1 (weight ratio, molar ratio, or w:v ratio), at least0.01:1, at least 0.01:1, at least 1:1, at least 2:1, at least 3:1, atleast 5:1, at least 10:1, at least 25:1, at least 50:1, at least 100:1,or at least 500:1. In some cases, the ratio of surface-altering agent topharmaceutical agent (or salt thereof) may be less than or equal to1000:1 (weight ratio or molar ratio), less than or equal to 500:1, lessthan or equal to 100:1, less than or equal to 75:1, less than or equalto 50:1, less than or equal to 25:1, less than or equal to 10:1, lessthan or equal to 5:1, less than or equal to 3:1, less than or equal to2:1, less than or equal to 1:1, or less than or equal to 0.1:1.Combinations of the above-referenced ranges are possible (e.g., a ratioof at least 5:1 and less than or equal to 50:1). Other ranges are alsopossible.

Surface-altering agents that may be suitable for use in coatingsinclude, for example, polymers and surfactants. Non-limiting examples ofpolymers that are suitable for use in coatings as surface-alteringagents, as described in more detail below, include poly(vinyl alcohol)and Pluronics®. Non-limiting examples of surfactants that are suitablefor use in coatings as surface-altering agents includeL-α-phosphatidylcholine (PC), 1,2-dipalmitoylphosphatidycholine (DPPC),oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitanmonolaurate, polyoxylene sorbitan fatty acid esters (Tweens),polysorbates (e.g., polyoxyethylene sorbitan monooleate) (e.g., Tween80®), polyoxyethylene sorbitan monostearate (e.g., Tween 60®),polyoxyethylene sorbitan monopalmitate (e.g., Tween 40®),polyoxyethylene sorbitan monolaurate (e.g., Tween 20®), naturallecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether,lauryl polyoxyethylene ether, polyoxylene alkyl ethers, block copolymersof oxyethylene and oxypropylene, polyoxyethylene sterates,polyoxyethylene castor oil and their derivatives, Vitamin-PEG and theirderivatives, synthetic lecithin, diethylene glycol dioleate,tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glycerylmonooleate, glyceryl monostearate, glyceryl monoricinoleate, cetylalcohol, stearyl alcohol, polyethylene glycol, cetyl pyridiniumchloride, benzalkonium chloride, olive oil, glyceryl monolaurate, cornoil, cotton seed oil, and sunflower seed oil. Derivatives of theabove-noted compounds are also possible. Combinations of the above-notedcompounds and others described herein may also be used assurface-altering agents in the inventive particles. As described herein,in some embodiments a surface-altering agent may act as a stabilizer, asurfactant, and/or an emulsifier. In some embodiments, the surfacealtering agent may aid particle transport in mucus.

It should be appreciated that while in some embodiments the stabilizerused for milling forms a coating on a particle surface, which coatingrenders particle mucus penetrating, in other embodiments, the stabilizermay be exchanged with one or more other surface-altering agents afterthe particle has been formed. For example, in one set of methods, afirst stabilizer/surface-altering agent may be used during a millingprocess and may coat a surface of a core particle, and then all orportions of the first stabilizer/surface-altering agent may be exchangedwith a second stabilizer/surface-altering agent to coat all or portionsof the core particle surface. In some cases, the secondstabilizer/surface-altering agent may render the particle mucuspenetrating more than the first stabilizer/surface-altering agent. Insome embodiments, a core particle having a coating including multiplesurface-altering agents may be formed.

Any suitable grinding medium can be used for milling. In someembodiments, a ceramic and/or polymeric material and/or a metal can beused. Examples of suitable materials may include zirconium oxide,silicon carbide, silicon oxide, silicon nitride, zirconium silicate,yttrium oxide, glass, alumina, alpha-alumina, aluminum oxide,polystyrene, poly(methyl methacrylate), titanium, steel. A grindingmedium may have any suitable size. For example, the grinding medium mayhave an average diameter of at least about 0.1 mm, at least about 0.2mm, at least about 0.5 mm, at least about 0.8 mm, at least about 1 mm,at least about 2 mm, or at least about 5 mm. In some cases, the grindingmedium may have an average diameter of less than or equal to about 5 mm,less than or equal to about 2 mm, less than or equal to about 1 mm, lessthan or equal to about 0.8, less than or equal to about 0.5 mm, or lessthan or equal to about 0.2 mm. Combinations of the above-referencedranges are also possible (e.g., an average diameter of at least about0.5 millimeters and less than or equal to about 1 mm). Other ranges arealso possible.

Any suitable solvent may be used for milling. The choice of solvent maydepend on factors such as the solid material (e.g., pharmaceuticalagent) being milled, the particular type of stabilizer/surface-alteringagent being used (e.g., one that may render the particle mucuspenetrating), the grinding material be used, among other factors.Suitable solvents may be ones that do not substantially dissolve thesolid material or the grinding material, but dissolve thestabilizer/surface-altering agent to a suitable degree. Non-limitingexamples of solvents may include water, buffered solutions, otheraqueous solutions, alcohols (e.g., ethanol, methanol, butanol), andmixtures thereof that may optionally include other components such aspharmaceutical excipients, polymers, pharmaceutical agents, salts,preservative agents, viscosity modifiers, buffering agents, tonicitymodifier, taste masking agents, antioxidants, pH modifier, and otherpharmaceutical excipients. In other embodiments, an organic solvent canbe used. A pharmaceutical agent may have any suitable solubility inthese or other solvents, such as a solubility in one or more of theranges described above for aqueous solubility or for solubility in acoating solution.

In one aspect, the invention provides a method of formingmucus-penetrating particles (MPP) using a core comprising apharmaceutical agent in the presence of other components, suchsurfactants or surface-altering agents, tonicity agents, salts,buffering agents, chelting agents, preservatives, diluents, and/orsolvents.

In another embodiment, the invention provides a method of forming MPPusing a core comprising loteprednol etabonate in the presence of othercomponents comprising a milling media, such surfactants orsurface-altering agents, tonicity agents, salts, buffering agents,chelting agents, preservatives, diluents, and/or solvents.

The method of forming the pharmaceutical agent mucus-penetratingparticles comprises a milling step and optionally, dilution step. In themilling step, a coarse aqueous suspension containing about 2-30%pharmaceutical agent is milled in the presence of milling media toproduce a nanosuspension of pharmaceutical agent particles sized in therange of about 200-500 nm. In another aspect, the particle size range isabout 200-300 nm. In a subsequent dilution step, the obtainednanocrystalline suspension separated from the milling media is mixed ina product vessel with post-milling diluent that can be the samecomponents used in the milling step in varying amounts, or othercombinations of components, such as surfactants or surface-alteringagents, tonicity agents, salts, buffering agents, chelting agents,preservatives, diluents, and/or solvents.

In one embodiment, the method comprises milling loteprednol etabonate inthe presence of milling media comprising a Pluronic or poloxamer (e.g.,poloxamer 407, poloxamer 338), glycerin, sodium chloride, disodiumedetate (disodium ethylenediaminetetraacetic acid (Na₂EDTA) or EDTA),and optionally, trisodium citrate, citric acid, and/or benzalkoniumchloride.

In other embodiments, core particles may be formed by an emulsificationtechnique (emulsification). Generally, emulsification techniques mayinvolve dissolving or dispersing a material to be used as the core in asolvent; this solution or dispersion is then emulsified in a secondimmiscible solvent, thereby forming a plurality of particles comprisingthe material. Suitable emulsification techniques may include formationof oil-in-water emulsions, water-in-oil emulsions, water-oil-wateremulsions, oil-water-oil emulsions, solid-in-oil-in-water emulsions, andsolid-in-water-in-oil emulsions, etc., with or without subsequentsolvent removal, for example, by evaporation or extraction.Emulsification techniques are versatile and may be useful for preparingcore particles comprising pharmaceutical agents having a relatively lowaqueous solubility as well as pharmaceutical agents having a relativelyhigh aqueous solubility.

In some embodiments, the core particles described herein may be producedby emulsification in the presence of one or more surface-alteringagents. In some such embodiments, the stabilizer may act as asurface-altering agent, forming a coating on the particle (i.e., theemulsification and coating steps may be performed substantiallysimultaneously).

In some embodiments, a method of forming a core particle byemulsification involves choosing a stabilizer that is suitable for bothemulsification and for forming a coating on the particle and renderingthe particle mucus penetrating. For example, as described in more detailbelow, it has been demonstrated that 200-500 nm nanoparticles of a modelpolymer PLA produced by emulsification in the presence of certain PVApolymers resulted in particles that can penetrate physiological mucussamples at the same rate as well-established PEGylated polymeric MPP.Interestingly, it was observed that only a subset of PVA polymers testedfit the criteria of being suitable for both emulsification and forforming a coating on the particle that renders the particle mucuspenetrating, as described in more detail below.

In other embodiments, the particles are first formed using anemulsification technique, following by coating of the particles with asurface-altering agent.

Any suitable solvent and solvent combinations can be used foremulsification. Some examples of solvents which can serve as oil phaseare organic solvents such chloroform, dichloromethane, ethyl acetate,ethyl ether, petroleum ether (hexane, heptane), and oils such as peanutoil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oilsoybean oil, and silicone oil. Some examples of solvents which can serveas water phase are water and aqueous buffers. Other solvents are alsopossible.

In other embodiments, core particles may be formed by a precipitationtechnique. Precipitation techniques (e.g., microprecipitationtechniques, nanoprecipitation techniques, crystallization techniques,controlled crystallization techniques) may involve forming a firstsolution comprising the material to be used as the core (e.g., apharmaceutical agent) and a solvent, wherein the material issubstantially soluble in the solvent. The solution may be added to asecond solution comprising another solvent in which the material issubstantially insoluble (i.e., an anti-solvent), thereby forming aplurality of particles comprising the material. In some cases, one ormore surface-altering agents, surfactants, materials, and/or bioactiveagents may be present in the first and/or second solutions. A coatingmay be formed during the process of precipitating the core (e.g., theprecipitating and coating steps may be performed substantiallysimultaneously). In other embodiments, the particles are first formedusing a precipitation technique, following by coating of the particleswith a surface-altering agent.

In some embodiments, a precipitation technique may be used to formpolymeric core particles with or without a pharmaceutical agent.Generally, a precipitation technique involves dissolving the polymer tobe used as the core in a solvent (with or without a pharmaceutical agentpresent), and the solution is then added to a miscible anti-solvent(with or without excipients present) to form the core particle. In someembodiments, this technique may be useful for preparing, for example,polymeric core particles comprising pharmaceutical agents that areslightly soluble (1-10 mg/L), very slightly soluble (0.1-1 mg/mL) orpractically insoluble (<0.1 mg/mL) in aqueous solutions (e.g., agentshaving a relatively low aqueous solubility).

Any suitable solvent can be used for precipitation. In some embodiments,a suitable solvent for precipitation may include, for example, acetone,acetonitrile, dimethylformamide, dimethysulfoxide,N-methyl-2-pyrrolidone, 2-pyrrolidone, tetrahydrofuran. Other organicsolvents and non-organic solvents can also be used.

Any suitable anti-solvent can be used for precipitation, including thesolvents described herein that may be used for milling. In one set ofembodiments, an aqueous solution is used (e.g., water, bufferedsolutions, other aqueous solutions, and alcohols such as ethanol,methanol, butanol), and mixtures thereof that may optionally includeother components such as pharmaceutical excipients, polymers, andpharmaceutical agents.

Surface-altering agents for emulsification and precipitation may bepolymers or surfactants, including the surface-altering agents describedherein that may be used for milling.

Non-limiting examples of suitable polymers suitable for forming all orportions of a core by emulsification or precipitation may includepolyamines, polyethers, polyamides, polyesters, polycarbamates,polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles,polyarylates, polypeptides, polynucleotides, and polysaccharides.Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D, L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropylmethacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid,poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(esteramides), polyamides, poly(ester ethers), polycarbonates, polyalkylenessuch as polyethylene and polypropylene, polyalkylene glycols such aspoly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkyleneterephthalates such as poly(ethylene terephthalate), polyvinyl alcohols(PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate),polyvinyl halides such as poly(vinyl chloride) (PVC),polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes,derivatized celluloses such as alkyl celluloses, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylicacids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointlyreferred to herein as “polyacrylic acids”), and copolymers and mixturesthereof, polydioxanone and its copolymers, polyhydroxyalkanoates,polypropylene fumarate), polyoxymethylene, poloxamers,poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone, bovine serum albumin, human serum albumin,collagen, DNA, RNA, carboxymethyl cellulose, chitosan, dextran.

Polymers suitable for forming all or portions of a core and/orsurface-altering agent may also include a poly(ethylene glycol)-vitaminE conjugate (hereinafter, “PEG-VitE conjugate”). The particles,compositions, and/or formulations including a PEG-VitE conjugate, andmethods of making and using the particles, compositions, and/orformulations, are provided in more detail in international PCTapplication publication WO2012/061703, which is incorporated herein byreference in its entirety for all purposes. In some cases, the molecularweight of the PEG portion of the PEG-VitE conjugate is greater thanabout 2 kDa. The molecular weight of the PEG portion of the PEG-VitEconjugate may be selected so as to aid in the formation and/or transportof the particle across a mucosal barrier as described herein. In someembodiments, use of a PEG-VitE conjugate with a PEG portion having amolecular weight greater than about 2 kDa may allow for greaterpenetration of the particles through a mucosal barrier as compared touse of a PEG-VitE conjugate with a PEG portion having a molecular weightless than about 2 kDa. Additionally, in certain embodiments a highermolecular weight PEG portion may facilitate drug encapsulation. Thecombined ability to act as a surfactant and to reduce mucoadhesionprovides important benefits as compared to other commonly usedsurfactants for drug encapsulation. In some cases, the molecular weightof the PEG portion of the PEG-VitE conjugate is between about 2 kDa andabout 8 kDa, or between about 3 kDa and about 7 kDa, or between about 4kDa and about 6 kDa, or between about 4.5 kDa and about 6.5 kDa, orabout 5 kDa.

In some embodiments, a precipitation technique may be used to formparticles comprised predominantly of a pharmaceutical agent (e.g.,nanocrystals). Generally, such a precipitation technique involvesdissolving the pharmaceutical agent to be used as the core in a solvent,which is then added to a miscible anti-solvent with or withoutexcipients to form the core particle. In some embodiments, thistechnique may be useful for preparing, for example, particles ofpharmaceutical agents that are slightly soluble (1-10 mg/L), veryslightly soluble (0.1-1 mg/mL) or practically insoluble (<0.1 mg/mL) inaqueous solutions (e.g., agents having a relatively low aqueoussolubility).

In some embodiments, precipitation by salt (or complex) formation may beused to form particles (e.g., nanocrystals) of a salt of apharmaceutical agent. Generally, precipitation by salt formationinvolves dissolving the material to be used as the core in a solventwith or without excipients followed by addition of a counter-ion or acomplexing agent, which forms an insoluble salt or a complex with thepharmaceutical agent to form the core particle. This technique may beuseful for preparing particles of pharmaceutical agents that are solublein aqueous solutions (e.g., agents having a relatively high aqueoussolubility). In some embodiments, pharmaceutical agents having one ormore charged or ionizable groups can interact with a counter-ion (e.g.,a cation or an anion) to form a salt complex.

A variety of counter-ions can be used to form salt complexes, includingmetals (e.g., alkali metals, alkali earth metals and transition metals).Non-limiting examples of cationic counter-ions include zinc, calcium,aluminum, zinc, barium, and magnesium. Non-limiting examples of anioniccounter-ions include phosphate, carbonate, and fatty acids. Counter-ionsmay be, for example, monovalent, divalent, or trivalent. Othercounter-ions are known in the art and can be used in the embodimentsdescribed herein. Other ionic and non-ionic complexing agents are alsopossible.

A variety of different acids may be used in a precipitation process. Insome embodiments, a suitable acid may include deconoic acid, hexanoicacid, mucic acid, octanoic acid. In other embodiments, a suitable acidmay include acetic acid, adipic acid, L-ascorbic acid, L-aspartic acid,capric acid (decanoic acid), carbonic acid, citric acid, fumaric acid,galactaric acid, D-glucoheptonic acid, D-gluconic acid, D-glucuronicacid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolicacid, hippuric acid, hydrochloric acid, DL-lactic acid, lauric acid,maleic acid, (−)-L-malic acid, palmitic acid, phosphoric acid, sebacicacid, stearic acid, succinic acid, sulfuric acid, (+)-L-tartaric acid,or thiocyanic acid. In other embodiments, a suitable acid may includealginic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid,caprylic acid (octanoic acid), cyclamic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, ethanesulfonic acid,2-hydroxy-, gentisic acid, glutaric acid, 2-oxo-, isobutyric acid,lactobionic acid, malonic acid, methanesulfonic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,2-naphthoic acid, 1-hydroxy-, nicotinic acid, oleic acid, orotic acid,oxalic acid, pamoic acid, (embonic acid), propionic acid,(−)-L-pyroglutamic acid, or p-toluenesulfonic acid. In yet otherembodiments, a suitable acid may include acetic acid, 2,2-dichloro-,benzoic acid, 4-acetamido-, (+)-camphor-10-sulfonic acid, caproic acid(hexanoic acid), cinnamic acid, formic acid, hydrobromic acid,DL-mandelic acid, nitric acid, salicylic acid, salicylic acid, 4-amino-,or undecylenic acid (undec-10-enoic acid). Mixtures of one or more suchacids can also be used.

A variety of different bases may be used in a precipitation process. Insome embodiments, a suitable base includes ammonia, L-arginine, calciumhydroxide, choline, glucamine, N-methyl-, lysine, magnesium hydroxide,potassium hydroxide, or sodium hydroxide. In other embodiments, asuitable base may include benethamine, benzathine, betaine, deanol,diethylamine, ethanol, 2-(diethylamino)-, hydrabamine, morpholine,4-(2-hydroxyethyl)-morpholine, pyrrolidine, 1-(2-hyroxyethyl)-, ortromethamine. In other embodiments, a suitable base may includediethanolamine (2,2′-iminobis(ethanol)), ethanolamine (2-aminoethanol),ethylenediamine, IH-imidazole, piperazine, triethanolamine(2,2′,2″-nitrilotris(ethanol)), or zinc hydroxide. Mixtures of one ormore such bases can also be used.

Any suitable solvent can be used for precipitation by salt formation,including the solvents described herein that may be used for milling. Inone set of embodiments, an aqueous solution is used (e.g., water,buffered solutions, other aqueous solutions, alcohols (e.g., ethanol,methanol, butanol), and mixtures thereof that may optionally includeother components such as pharmaceutical excipients, polymers, andpharmaceutical agents.

In the precipitation process, the salt may have a lower aqueoussolubility (or solubility in the solvent containing the salt) than thepharmaceutical agent in the non-salt form. The aqueous solubility (orsolubility in the solvent) of the salt may be, for example, less than orequal to about 5 mg/mL, less than or equal to about 2 mg/mL, less thanor equal to about 1 mg/mL, less than or equal to about 0.5 mg/mL, lessthan or equal to about 0.1 mg/mL, less than or equal to about 0.05mg/mL, or less than or equal to about 0.01 mg/mL, less than or equal toabout 1 μg/mL, less than or equal to about 0.1 μg/mL, less than or equalto about 0.01 μg/mL, less than or equal to about 1 ng/mL, less than orequal to about 0.1 ng/mL, or less than or equal to about 0.01 ng/mL at25° C. In some embodiments, the salt may have an aqueous solubility (orsolubility in the solvent) of at least about 1 μg/mL, at least about 10μg/mL, at least about 0.1 ng/mL, at least about 1 ng/mL, at least about10 ng/mL, at least about 0.1 μg/mL, at least about 1 μg/mL, at leastabout 5 μg/mL, at least about 0.01 mg/mL, at least about 0.05 mg/mL, atleast about 0.1 mg/mL, at least about 0.5 mg/mL, at least about 1.0mg/mL, at least about 2 mg/mL. Combinations of the above-noted rangesare possible (e.g., an aqueous solubility (or solubility in the solvent)of at least about 0.001 mg/mL and less than or equal to about 1 mg/mL).Other ranges are also possible. The salt may have these or other rangesof aqueous solubilities at any point throughout the pH range (e.g., frompH 1 to pH 14).

In some embodiments, the solvent used for precipitation includes one ormore surface-altering agents as described herein, and a coating of theone or more surface-altering agents may be formed around the particle asit precipitates out of solution. The surface-altering agent may bepresent in the solvent at any suitable concentration, such as aconcentration of at least about 0.001% (w/v), at least about 0.005%(w/v), at least about 0.01% (w/v), at least about 0.05% (w/v), at leastabout 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), orat least about 5% (w/v) in the aqueous solution. In some instances, thesurface-altering agent is present in the solvent at a concentration ofless than or equal to about 5% (w/v), less than or equal to about 1%(w/v), less than or equal to about 0.5% (w/v), less than or equal toabout 0.1% (w/v), less than or equal to about 0.05% (w/v), less than orequal to about 0.01% (w/v), or less than or equal to about 0.005% (w/v).Combinations of the above-referenced ranges are also possible (e.g., aconcentration of at least about 0.01 (w/v) and less than or equal toabout 1% (w/v). Other ranges are also possible.

Another exemplary method of forming a core particle includes afreeze-drying technique. In this technique, a pharmaceutical agent orsalt thereof may be dissolved in an aqueous solution, optionallycontaining a surface-altering agent. A counter-ion may be added to thesolution, and the solution may be immediately flash frozen and freezedried. Dry powder can be reconstituted in a suitable solvent (e.g., anaqueous solution such as water) at a desired concentration.

A counter-ion may be added to a solvent for freeze-drying in anysuitable range. In some cases, the ratio of counter-ion topharmaceutical agent (e.g., salt) may be at least 0.1:1 (weight ratio ormolar ratio), at least 1:1, at least 2:1, at least 3:1, at least 5:1, atleast 10:1, at least 25:1, at least 50:1, or at least 100:1. In somecases, the ratio of counter-ion to pharmaceutical agent (e.g., salt) maybe less than or equal to 100:1 (weight ratio or molar ratio), less thanor equal to 75:1, less than or equal to 50:1, less than or equal to25:1, less than or equal to 10:1, less than or equal to 5:1, less thanor equal to 3:1, less than or equal to 2:1, less than or equal to 1:1,or less than or equal to 0.1:1. Combinations of the above-referencedranges are possible (e.g., a ratio of at least 5:1 and less than orequal to 50:1). Other ranges are also possible.

If the surface-altering agent is present in the solvent prior to freezedrying, it may be present at any suitable concentration, such as aconcentration of at least about 0.001% (w/v), at least about 0.005%(w/v), at least about 0.01% (w/v), at least about 0.05% (w/v), at leastabout 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), orat least about 5% (w/v) in the aqueous solution. In some instances, thesurface-altering agent is present in the solvent at a concentration ofless than or equal to about 5% (w/v), less than or equal to about 1%(w/v), less than or equal to about 0.5% (w/v), less than or equal toabout 0.1% (w/v), less than or equal to about 0.05% (w/v), less than orequal to about 0.01% (w/v), or less than or equal to about 0.005% (w/v).Combinations of the above-referenced ranges are also possible (e.g., aconcentration of at least about 0.01% (w/v) and less than or equal toabout 1% (w/v). Other ranges are also possible.

The concentration of surface-altering agent present in the solvent maybe above or below the critical micelle concentration (CMC) of thesurface-altering agent, depending on the particular surface-alteringagent used. In other embodiments, stable particles can be formed byadding excess counter-ion to a solution containing a pharmaceuticalagent. The precipitate can then be washed by various methods such ascentrifugation. The resultant slurry may be sonicated. One or moresurface-altering agents may be added to stabilize the resultantparticles.

Other methods of forming core particles are also possible. Techniquesfor forming core particles may include, for example, coacervation-phaseseparation; melt dispersion; interfacial deposition; in situpolymerization; self-assembly of macromolecules (e.g., formation ofpolyelectrolyte complexes or polyelectrolyte-surfactant complexes);spray-drying and spray-congealing; electro-spray; air suspensioncoating; pan and spray coating; freeze-drying, air drying, vacuumdrying, fluidized-bed drying; precipitation (e.g., nanoprecipitation,microprecipitation); critical fluid extraction; and lithographicapproaches (e.g., soft lithography, step and flash imprint lithography,interference lithography, photolithography).

Combinations of the methods described herein and other methods are alsopossible. For example, in some embodiments, a core of a pharmaceuticalagent is first formed by precipitation, and then the size of the core isfurther reduced by a milling process.

Following formation of particles of a pharmaceutical agent, theparticles may be optionally exposed to a solution comprising a (second)surface-altering agent that may associate with and/or coat theparticles. In embodiments in which the pharmaceutical agent alreadyincludes a coating of a first surface-altering agent, all or portions ofa second surface-altering agent may be exchanged with a secondstabilizer/surface-altering agent to coat all or portions of theparticle surface. In some cases, the second surface-altering agent mayrender the particle mucus penetrating more than the firstsurface-altering agent. In other embodiments, a particle having acoating including multiple surface-altering agents may be formed (e.g.,in a single layer or in multiple layers). In other embodiments, aparticle having multiple coatings (e.g., each coating optionallycomprising different surface-altering agents) may be formed. In somecases, the coating is in the form of a monolayer of a surface-alteringagent. Other configurations are also possible.

In any of the methods described herein, a particle may be coated with asurface-altering agent by incubating the particle in a solution with thesurface-altering agent for a period of at least about 1 minutes, atleast about 2 minutes, at least about 5 min., at least about 10 min., atleast about 15 min., at least about 20 min., at least about 30 min., atleast about 60 min., or more. In some cases, incubation may take placefor a period of less than or equal to about 10 hours, less than or equalto about 5 hours, or less than or equal to about 60 min. Combinations ofthe above referenced ranges are also possible (e.g., an incubationperiod of less than or equal to 60 min. and at least about 2 min.).

Particle Coatings

As shown in the embodiment illustrated in FIG. 1, core 16 may besurrounded by coating 20 comprising one or more surface-altering agents.In some embodiments, the coating is formed of one or moresurface-altering agents or other molecules disposed on the surface ofthe core. The particular chemical makeup and/or components of thecoating and surface-altering agent(s) can be chosen so as to impartcertain functionality to the particles, such as enhanced transportthrough mucosal barriers.

It should be understood that a coating which surrounds a core need notcompletely surround the core, although such embodiments may be possible.For example, the coating may surround at least about 10%, at least about30%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 99% of thesurface area of a core. In some cases, the coating substantiallysurrounds a core. In other cases, the coating completely surrounds acore. In other embodiments, a coating surrounds less than or equal toabout 100%, less than or equal to about 90%, less than or equal to about80%, less than or equal to about 70%, less than or equal to about 60%,or less than or equal to about 50% of the surface area of a core.Combinations of the above-referenced ranges are also possible (e.g.,surrounding greater than 80% and less than 100% of the surface area of acore).

The components of the coating may be distributed evenly across a surfaceof the core in some cases, and unevenly in other cases. For example, thecoating may include portions (e.g., holes) that do not include anymaterial in some cases. If desired, the coating may be designed to allowpenetration and/or transport of certain molecules and components into orout of the coating, but may prevent penetration and/or transport ofother molecules and components into or out of the coating. The abilityof certain molecules to penetrate and/or be transported into and/oracross a coating may depend on, for example, the packing density of thesurface-altering agents forming the coating and the chemical andphysical properties of the components forming the coating. As describedherein, the coating may include one layer of material (e.g., amonolayer), or multilayers of materials in some embodiments. A singletype of surface-altering agent may be present, or multiple types ofsurface-altering agent.

A coating of a particle can have any suitable thickness. For example, acoating may have an average thickness of at least about 1 nm, at leastabout 5 nm, at least about 10 nm, at least about 30 nm, at least about50 nm, at least about 100 nm, at least about 200 nm, at least about 500nm, at least about 1 μm, or at least about 5 μm. In some cases, theaverage thickness of a coating is less than or equal to about 5 μm, lessthan or equal to about 1 μm, less than or equal to about 500 nm, lessthan or equal to about 200 nm, less than or equal to about 100 nm, lessthan a to about 50 nm, less than or equal to about 30 nm, less than orequal to about 10 nm, or less than or equal to about 5 nm. Combinationsof the above-referenced ranges are also possible (e.g., an averagethickness of at least about 1 nm and less than or equal to about 100nm). Other ranges are also possible. For particles having multiplecoatings, each coating layer may have one of the thicknesses describedabove.

In some embodiments, the compositions and methods described herein mayallow for the coating of a core particle with hydrophilicsurface-altering moieties without requiring covalent linking of thesurface-altering moieties to the core surface. In some such embodiments,a core having a hydrophobic surface may be coated with a polymerdescribed herein, thereby causing a plurality of surface-alteringmoieties to be on the core surface without substantially altering thecharacteristics of the core itself. For example, the surface alteringagent may be adsorbed to the outer surface of the core particle. Inother embodiments, however, a surface-altering agent is covalentlylinked to a core particle.

In certain embodiments in which the surface-altering agent is adsorbedonto a surface of a core, the surface-altering agent may be inequilibrium with other molecules of the surface-altering agent insolution, optionally with other components (e.g., in acomposition/formulation). In some cases, the adsorbed surface-alteringagent may be present on the surface of the core at a density describedherein. The density may be an average density as the surface alteringagent is in equilibrium with other components in solution.

The coating and/or surface-altering agent of a particle described hereinmay comprise any suitable material, such as a hydrophobic material, ahydrophilic material, and/or an amphiphilic material. In someembodiments, the coating includes a polymer. In certain embodiments, thepolymer is a synthetic polymer (i.e., a polymer not produced in nature).In other embodiments, the polymer is a natural polymer (e.g., a protein,polysaccharide, rubber). In certain embodiments, the polymer is asurface active polymer. In certain embodiments, the polymer is anon-ionic polymer. In certain embodiments, the polymer is a linear,synthetic non-ionic polymer. In certain embodiments, the polymer is anon-ionic block copolymer. In some embodiments, the polymer may be acopolymer, e.g., where one repeat unit is relatively hydrophobic andanother repeat unit is relatively hydrophilic. The copolymer may be, forexample, a diblock, triblock, alternating, or random copolymer. Thepolymer may be charged or uncharged.

In some embodiments, a coating comprises a synthetic polymer havingpendant hydroxyl groups on the backbone of the polymer. For example, incertain embodiments, the polymer may include poly(vinyl alcohol), apartially hydrolyzed poly(vinyl acetate) or a copolymer of vinyl alcoholand vinyl acetate. In certain embodiments, a synthetic polymer havingpendant hydroxyl groups on the backbone of the polymer may includepoly(ethylene glycol)-poly(vinyl acetate)-poly(vinyl alcohol)copolymers, poly(ethylene glycol)-poly(vinyl alcohol) copolymers,poly(propylene oxide)-poly(vinyl alcohol) copolymers, and poly(vinylalcohol)-poly(acryl amide) copolymers. Without wishing to be bound bytheory, a particle including a coating comprising a synthetic polymerhaving pendant hydroxyl groups on the backbone of the polymer may havereduced mucoadhesion as compared to a control particle due to, at leastin part, the display of a plurality of hydroxyl groups on the particlesurface. One possible mechanism for the reduced mucoadhesion is that thehydroxyl groups alter the microenvironment of the particle, for example,by ordering water and other molecules in the particle/mucus environment.An additional or alternative possible mechanism is that the hydroxylgroups shield the adhesive domains of the mucin fibers, thereby reducingparticle adhesion and speeding up particle transport.

Moreover, the ability of a particle coated with a synthetic polymerhaving pendant hydroxyl groups on the backbone of the polymer to bemucus penetrating may also depend, at least in part, on the degree ofhydrolysis of the polymer. In some embodiments, the hydrophobic portionsof the polymer (e.g., portions of the polymer that are not hydrolyzed)may allow the polymer to be adhered to the core surface (e.g., in thecase of the core surface being hydrophobic), thus allowing for a strongassociation between the core and the polymer. Surprisingly, it has beenfound that in some embodiments involving the surface-altering agent PVA,too high of a degree of hydrolysis does not allow for sufficientadhesion between the PVA and the core (e.g., in the case of the corebeing hydrophobic), and thus, the particles coated with such a polymergenerally do not exhibit sufficient reduced mucoadhesion. In someembodiments, too low of a degree of hydrolysis does not enhance particletransport in mucus, perhaps due to the lower amounts of hydroxyl groupsavailable for altering the microenvironment of the particle and/orshielding the adhesive domains of the mucin fibers.

A synthetic polymer having pendant hydroxyl groups on the backbone ofthe polymer may have any suitable degree of hydrolysis (and, therefore,varying amounts of hydroxyl groups). The appropriate level of hydrolysismay depend on additional factors such as the molecular weight of thepolymer, the composition of the core, the hydrophobicity of the core,etc. In some embodiments, a synthetic polymer (e.g., PVA or partiallyhydrolyzed poly(vinyl acetate) or a copolymer of vinyl alcohol and vinylacetate) may be at least about 30% hydrolyzed, at least about 35%hydrolyzed, at least about 40% hydrolyzed, at least about 45%hydrolyzed, at least about 50% hydrolyzed, at least about 55%hydrolyzed, at least about 60% hydrolyzed, at least about 65%hydrolyzed, at least about 70% hydrolyzed, at least about 75%hydrolyzed, at least about 80% hydrolyzed, at least about 85%hydrolyzed, at least about 87% hydrolyzed, at least about 90%hydrolyzed, at least about 95% hydrolyzed, or at least about 98%hydrolyzed. In some embodiments, the synthetic polymer may be less thanor equal to about 100% hydrolyzed, less than or equal to about 98%hydrolyzed, less than or equal to about 97% hydrolyzed, less than orequal to about 96% hydrolyzed, less than or equal to about 95%hydrolyzed, less than or equal to about 94% hydrolyzed, less than orequal to about 93% hydrolyzed, less than or equal to about 92%hydrolyzed, less than or equal to about 91% hydrolyzed, less than orequal to about 90% hydrolyzed, less than or equal to about 87%hydrolyzed, less than or equal to about 85% hydrolyzed, less than orequal to about 80% hydrolyzed, less than or equal to about 75%hydrolyzed, less than or equal to about 70% hydrolyzed, or less than orequal to about 60% hydrolyzed. Combinations of the above-mentionedranges are also possible (e.g., a polymer that is at least about 80%hydrolyzed and less than or equal to about 95% hydrolyzed). Other rangesare also possible.

The molecular weight of a synthetic polymer described herein (e.g., onehaving pendant hydroxyl groups on the backbone of the polymer) may beselected so as to reduce the mucoadhesion of a core and to ensuresufficient association of the polymer with the core. In certainembodiments, the molecular weight of the synthetic polymer is at leastabout 1 kDa, at least about 2 kDa, at least about 5 kDa, at least about8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 12kDa, at least about 15 kDa at least about 20 kDa, at least about 25 kDa,at least about 30 kDa, at least about 40 kDa, at least about 50 kDa, atleast about 60 kDa, at least about 70 kDa, at least about 80 kDa, atleast about 90 kDa, at least about 100 kDa at least about 110 kDa, atleast about 120 kDa, at least about 130 kDa, at least about 140 kDa, atleast about 150 kDa, at least about 200 kDa, at least about 500 kDa, orat least about 1000 kDa. In some embodiments, the molecular weight ofthe synthetic polymer is less than or equal to about 1000 kDa, less thanor equal to about 500 kDa, less than or equal to about 200 kDa, lessthan or equal to about 180 kDa, less than or equal to about 150 kDa,less than or equal to about 130 kDa, less than or equal to about 120kDa, less than or equal to about 100 kDa, less than or equal to about 85kDa, less than or equal to about 70 kDa, less than or equal to about 65kDa, less than or equal to about 60 kDa, less than or equal to about 50kDa, or less than or equal to about 40 kDa, less than or equal to about30 kDa, less than or equal to about 20 kDa, less than or equal to about15 kDa, or less than or equal to about 10 kDa. Combinations of theabove-mentioned ranges are also possible (e.g., a molecular weight of atleast about 10 kDa and less than or equal to about 30 kDa). Theabove-mentioned molecular weight ranges can also be combined with theabove-mentioned hydrolysis ranges to form suitable polymers.

In some embodiments, a synthetic polymer described herein is orcomprises PVA. PVA is a non-ionic polymer with surface activeproperties. It is a synthetic polymer typically produced throughhydrolysis of poly(vinyl acetate). Partially hydrolyzed PVA is comprisedof two types of repeating units: vinyl alcohol units and residual vinylacetate units. The vinyl alcohol units are relatively hydrophilic; thevinyl acetate units are relatively hydrophobic. In some instances, thesequence distribution of vinyl alcohol units and vinyl acetate units isblocky. For example, a series of vinyl alcohol units may be followed bya series of vinyl acetate units, and followed by more vinyl alcoholunits to form a polymer having a mixed block-copolymer type arrangement,with units distributed in a blocky manner. In certain embodiments, therepeat units form a copolymer, e.g., a diblock, triblock, alternating,or random copolymer. Polymers other than PVA may also have theseconfigurations of hydrophilic units and hydrophobic units.

In some embodiments, the hydrophilic units of a synthetic polymerdescribed herein may be substantially present at the outer surface ofthe particle. For example, the hydrophilic units may form a majority ofthe outer surface of the coating and may help stabilize the particle inan aqueous solution containing the particle. The hydrophobic units maybe substantially present in the interior of the coating and/or at thesurface of the core particle, e.g., to facilitate attachment of thecoating to the core.

The molar fraction of the relatively hydrophilic units and therelatively hydrophobic units of a synthetic polymer may be selected soas to reduce the mucoadhesion of a core and to ensure sufficientassociation of the polymer with the core, respectively. As describedherein, the molar fraction of the hydrophobic units of the polymer maybe chosen such that adequate association of the polymer with the coreoccurs, thereby increasing the likelihood that the polymer remainsadhered to the core. The molar fraction of the relatively hydrophilicunits to the relatively hydrophobic units of a synthetic polymer may be,for example, at least 0.5:1, at least 1:1, at least 2:1, at least 3:1,at least 5:1, at least 7:1, at least 10:1, at least 15:1, at least 20:1,at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least75:1, or at least 100:1. In some embodiments, the molar fraction of therelatively hydrophilic units to the relatively hydrophobic units of asynthetic polymer may be, for example, less than or equal to 100:1, lessthan or equal to 75:1, less than or equal to 50:1, less than or equal to40:1, less than or equal to 30:1, less than or equal to 25:1, less thanor equal to 20:1, less than or equal to 15:1, less than or equal to10:1, less than or equal to 7:1, less than or equal to 5:1, less than orequal to 3:1, less than or equal to 2:1, or less than or equal to 1:1.Combinations of the above-referenced ranges are also possible (e.g., aratio of at least 1:1 and less than or equal to 50:1). Other ranges arealso possible.

The molecular weight of the PVA polymer may also be tailored to increasethe effectiveness of the polymer to render particles mucus penetrating.Examples of PVA polymers having various molecular weights and degree ofhydrolysis are shown in Table 1.

TABLE 1 Grades of PVA. The molecular weight (MW) and hydrolysis degreevalues were provided by the manufacturers. PVA acronym* MW, kDaHydrolysis degree, % 2K75  2 75-79 9K80  9-10 80 13K87 13-23 87-89 13K9813-23 98 31K87 31-50 87-89 31K98 31-50 98-99 57K86 57-60 86-89 85K87 85-124 87-89 85K99  85-124  99+ 95K95  95 95 105K80 104 80 130K87 13087-89 *PVA acronym explanation: XXKYY, where XX stands for the PVA'slower-end molecular weight in kDa and YY stands for the PVA's lower-endhydrolysis in %.

In certain embodiments, the synthetic polymer is represented by theformula:

wherein n is an integer between 0 and 22730, inclusive; and m is aninteger between 0 and 11630, inclusive. In certain embodiments, n is aninteger between 25 and 20600, inclusive. In some embodiments, m is aninteger between 5 and 1100, inclusive. In certain embodiments, m is aninteger between 0 and 400 inclusive or between 1 and 400 inclusive. Itis noted that n and m represent the total content of the vinyl alcoholand vinyl acetate repeat units in the polymer, respectively, rather thanthe block lengths.

The value of n may vary. In certain embodiments, n is at least 5, atleast 10, at least 20, at least 30, at least 50, at least 100, at least200, at least 300, at least 500, at least 800, at least 1000, at least1200, at least 1500, at least 1800, at least 2000, at least 2200, atleast 2400, at least 2600, at least 3000, at least 5000, at least 10000,at least 15000, at least 20000, or at least 25000. In some cases, n isless than or equal to 30000, less than or equal to 25000, less than orequal to 20000, less than or equal to 25000, less than or equal to20000, less than or equal to 15000, less than or equal to 10000, lessthan or equal to 5000, less than or equal to 3000, less than or equal to2800, less than or equal to 2400, less than or equal to 2000, less thanor equal to 1800, less than or equal to 1500, less than or equal to1200, less than or equal to 1000, less than or equal to 800, less thanor equal to 500, less than or equal to 300, less than or equal to 200,less than or equal to 100, or less than or equal to 50. Combinations ofthe above-referenced ranges are also possible (e.g., n being at least 50and less than or equal to 2000). Other ranges are also possible.

Similarly, the value of m may vary. For instance, in certainembodiments, m is at least 5, at least 10, at least 20, at least 30, atleast 50, at least 70, at least 100, at least 150, at least 200, atleast 250, at least 300, at least 350, at least 400, at least 500, atleast 800, at least 1000, at least 1200, at least 1500, at least 1800,at least 2000, at least 2200, at least 2400, at least 2600, at least3000, at least 5000, at least 10000, or at least 15000. In some cases, mis less than or equal to 15000, less than or equal to 10000, less thanor equal to 5000, less than or equal to 3000, less than or equal to2800, less than or equal to 2400, less than or equal to 2000, less thanor equal to 1800, less than or equal to 1500, less than or equal to1200, less than or equal to 1000, less than or equal to 800, less thanor equal to 500, less than or equal to 400, less than or equal to 350,less than or equal to 300, less than or equal to 250, less than or equalto 200, less than or equal to 150, less than or equal to 100, less thanor equal to 70, less than or equal to 50, less than or equal to 30, lessthan or equal to 20, or less than or equal to 10. Combinations of theabove-referenced ranges are also possible (e.g., m being at least 5 andless than or equal to 200). Other ranges are also possible.

In some embodiments, the particles described herein include a coatingcomprising a block copolymer having a relatively hydrophilic block and arelatively hydrophobic block. In some cases, the hydrophilic blocks maybe substantially present at the outer surface of the particle. Forexample, the hydrophilic blocks may form a majority of the outer surfaceof the coating and may help stabilize the particle in an aqueoussolution containing the particle. The hydrophobic block may besubstantially present in the interior of the coating and/or at thesurface of the core particle, e.g., to facilitate attachment of thecoating to the core. In some instances, the coating comprises asurface-altering agent including a triblock copolymer, wherein thetriblock copolymer comprises a hydrophilic block-hydrophobicblock-hydrophilic block configuration. Diblock copolymers having ahydrophilic block-hydrophobic block configuration are also possible.Combinations of block copolymers with other polymers suitable for use ascoatings are also possible. Non-linear block configurations are alsopossible such as in comb, brush, or star copolymers. In someembodiments, the relatively hydrophilic block includes a syntheticpolymer having pendant hydroxyl groups on the backbone of the polymer(e.g., PVA).

The molecular weight of the hydrophilic blocks and the hydrophobicblocks of the block copolymers may be selected so as to reduce themucoadhesion of a core and to ensure sufficient association of the blockcopolymer with the core, respectively. The molecular weight of thehydrophobic block of the block copolymer may be chosen such thatadequate association of the block copolymer with the core occurs,thereby increasing the likelihood that the block copolymer remainsadhered to the core.

In certain embodiments, the combined molecular weight of the (one ormore) relatively hydrophobic blocks or repeat units of a block copolymeris at least about 0.5 kDa, at least about 1 kDa, at least about 2 kDa,at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, atleast about 6 kDa, at least about 10 kDa, at least about 12 kDa, atleast about 15 kDa, at least about 20 kDa, or at least about 50 kDa, atleast about 60 kDa, at least about 70 kDa, at least about 80 kDa, atleast about 90 kDa, at least about 100 kDa at least about 110 kDa, atleast about 120 kDa, at least about 130 kDa, at least about 140 kDa, atleast about 150 kDa, at least about 200 kDa, at least about 500 kDa, orat least about 1000 kDa. In some embodiments, the combined molecularweight of the (one or more) relatively hydrophobic blocks or repeatunits is less than or equal to about 1000 kDa, less than or equal toabout 500 kDa, less than or equal to about 200 kDa, less than or equalto about 150 kDa, less than or equal to about 140 kDa, less than orequal to about 130 kDa, less than or equal to about 120 kDa, less thanor equal to about 110 kDa, less than or equal to about 100 kDa, lessthan or equal to about 90 kDa, less than or equal to about 80 kDa, lessthan or equal to about 50 kDa, less than or equal to about 20 kDa, lessthan or equal to about 15 kDa, less than or equal to about 13 kDa, lessthan or equal to about 12 kDa, less than or equal to about 10 kDa, lessthan or equal to about 8 kDa, or less than or equal to about 6 kDa.Combinations of the above-mentioned ranges are also possible (e.g., atleast about 3 kDa and less than or equal to about 15 kDa). Other rangesare also possible.

In some embodiments, the combined (one or more) relatively hydrophilicblocks or repeat units of a block copolymer constitute at least about 15wt %, at least about 20 wt %, at least about 25 wt %, at least about 30wt %, at least about 35 wt %, at least about 40 wt %, at least about 45wt %, at least about 50 wt %, at least about 55 wt %, at least about 60wt %, at least about 65 wt %, or at least about 70 wt % of the blockcopolymer. In some embodiments, the combined (one or more) relativelyhydrophilic blocks or repeat units of a block copolymer constitute lessthan or equal to about 90 wt %, less than or equal to about 80 wt %,less than or equal to about 60 wt %, less than or equal to about 50 wt%, or less than or equal to about 40 wt % of the block copolymer.Combinations of the above-referenced ranges are also possible (e.g., atleast about 30 wt % and less than or equal to about 80 wt %). Otherranges are also possible.

In some embodiments, the combined molecular weight of the (one or more)relatively hydrophilic blocks or repeat units of the block copolymer maybe at least about 0.5 kDa, at least about 1 kDa, at least about 2 kDa,at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, atleast about 6 kDa, at least about 10 kDa, at least about 12 kDa, atleast about 15 kDa, at least about 20 kDa, or at least about 50 kDa, atleast about 60 kDa, at least about 70 kDa, at least about 80 kDa, atleast about 90 kDa, at least about 100 kDa at least about 110 kDa, atleast about 120 kDa, at least about 130 kDa, at least about 140 kDa, atleast about 150 kDa, at least about 200 kDa, at least about 500 kDa, orat least about 1000 kDa. In certain embodiments, the combined molecularweight of the (one or more) relatively hydrophilic blocks or repeatunits is less than or equal to about 1000 kDa, less than or equal toabout 500 kDa, less than or equal to about 200 kDa, less than or equalto about 150 kDa, less than or equal to about 140 kDa, less than orequal to about 130 kDa, less than or equal to about 120 kDa, less thanor equal to about 110 kDa, less than or equal to about 100 kDa, lessthan or equal to about 90 kDa, less than or equal to about 80 kDa, lessthan or equal to about 50 kDa, less than or equal to about 20 kDa, lessthan or equal to about 15 kDa, less than or equal to about 13 kDa, lessthan or equal to about 12 kDa, less than or equal to about 10 kDa, lessthan or equal to about 8 kDa, less than or equal to about 6 kDa, lessthan or equal to about 5 kDa, less than or equal to about 3 kDa, lessthan or equal to about 2 kDa, or less than or equal to about 1 kDa.Combinations of the above-mentioned ranges are also possible (e.g., atleast about 0.5 kDa and less than or equal to about 3 kDa). Other rangesare also possible. In embodiments in which two hydrophilic blocks flanka hydrophobic block, the molecular weights of the two hydrophilic blocksmay be substantially the same or different.

In certain embodiments, the polymer of a surface-altering agent includesa polyether portion. In certain embodiments, the polymer includes apolyalkylether portion. In certain embodiments, the polymer includespolyethylene glycol tails. In certain embodiments, the polymer includesa polypropylene glycol central portion. In certain embodiments, thepolymer includes polybutylene glycol as the central portion. In certainembodiments, the polymer includes polypentylene glycol as the centralportion. In certain embodiments, the polymer includes polyhexyleneglycol as the central portion. In certain embodiments, the polymer is adiblock copolymer of one of the polymers described herein. In certainembodiments, the polymer is a triblock copolymer of one of the polymersdescribed herein. As disclosed herein, any recitation of PEG may bereplaced with polyethylene oxide (PEO), and any recitation of PEO may bereplaced with PEG. In some embodiments, a diblock or triblock copolymercomprises a synthetic polymer having pendant hydroxyl groups on thebackbone of the polymer (e.g., PVA) as one or more of the blocks (withvarying degrees of hydrolysis and varying molecular weights as describedherein). The synthetic polymer blocks may form the central portion orthe end portions of the block copolymer.

In certain embodiments, the polymer is a triblock copolymer of apolyalkyl ether (e.g., polyethylene glycol, polypropylene glycol) andanother polymer (e.g., a synthetic polymer having pendant hydroxylgroups on the backbone of the polymer (e.g., PVA). In certainembodiments, the polymer is a triblock copolymer of a polyalkyl etherand another polyalkyl ether. In certain embodiments, the polymer is atriblock copolymer of polyethylene glycol and another polyalkyl ether.In certain embodiments, the polymer is a triblock copolymer ofpolypropylene glycol and another polyalkyl ether. In certainembodiments, the polymer is a triblock copolymer with at least one unitof polyalkyl ether. In certain embodiments, the polymer is a triblockcopolymer of two different polyalkyl ethers. In certain embodiments, thepolymer is a triblock copolymer including a polyethylene glycol unit. Incertain embodiments, the polymer is a triblock copolymer including apolypropylene glycol unit. In certain embodiments, the polymer is atriblock copolymer of a more hydrophobic unit flanked by two morehydrophilic units. In certain embodiments, the hydrophilic units are thesame type of polymer. In some embodiments, the hydrophilic units includea synthetic polymer having pendant hydroxyl groups on the backbone ofthe polymer (e.g., PVA). In certain embodiments, the polymer includes apolypropylene glycol unit flanked by two more hydrophilic units. Incertain embodiments, the polymer includes two polyethylene glycol unitsflanking a more hydrophobic unit. In certain embodiments, the polymer isa triblock copolymer with a polypropylene glycol unit flanked by twopolyethylene glycol units. The molecular weights of the two blocksflanking the central block may be substantially the same or different.

In certain embodiments, the polymer is of the formula:

wherein n is an integer between 2 and 1140, inclusive; and m is aninteger between 2 and 1730, inclusive. In certain embodiments, n is aninteger between 10 and 170, inclusive. In certain embodiments, m is aninteger between 5 and 70 inclusive. In certain embodiments, n is atleast 2 times m, 3 times m, or 4 times m.

In certain embodiments, the coating includes a surface-altering agentcomprising a (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (hereinafter“PEG-PPO-PEG triblock copolymer”), present in the coating alone or incombination with another polymer such as a synthetic polymer havingpendant hydroxyl groups on the backbone of the polymer (e.g., PVA). Asdescribed herein, the PEG blocks may be interchanged with PEO blocks insome embodiments. The molecular weights of the PEG (or PEO) and PPOsegments of the PEG-PPO-PEG triblock copolymer may be selected so as toreduce the mucoadhesion of the particle, as described herein. Withoutwishing to be bound by theory, a particle having a coating comprising aPEG-PPO-PEG triblock copolymer may have reduced mucoadhesion as comparedto a control particle due to, at least in part, the display of aplurality of PEG (or PEO) segments on the particle surface. The PPOsegment may be adhered to the core surface (e.g., in the case of thecore surface being hydrophobic), thus allowing for a strong associationbetween the core and the triblock copolymer. In some cases, thePEG-PPO-PEG triblock copolymer is associated with the core throughnon-covalent interactions. For purposes of comparison, the controlparticle may be, for example, a carboxylate-modified polystyreneparticle of similar size as the coated particle in question.

In certain embodiments, a surface-altering agent includes a polymercomprising a poloxamer, having the trade name Pluronic®. Pluronic®polymers that may be useful in the embodiments described herein include,but are not limited to, F127, F38, F108, F68, F77, F87, F88, F98, L101,L121, L31, L35, L43, L44, L61, L62, L64, L81, L92, N3, P103, P104, P105,P123, P65, P84, and P85.

Examples of molecular weights of certain Pluronic® molecules are shownin Table 2.

TABLE 2 Molecular Weights of Pluronic ® molecules MW Pluronic ® AverageMW MW PPO PEO wt % PEO L31 1000 900 10 100 L44 2000 1200 40 800 L81 26672400 10 267 L101 3333 3000 10 333 P65 3600 1800 50 1800 L121 4000 360010 400 P103 4286 3000 30 1286 F38 4500 900 80 3600 P123 5143 3600 301543 P105 6000 3000 50 3000 F87 8000 2400 70 5600 F68 9000 1800 80 7200F127 12000 3600 70 8400 P123 5750 4030 30 1730

Although other ranges may be possible and useful in certain embodimentsdescribed herein, in some embodiments, the hydrophobic block of thePEG-PPO-PEG triblock copolymer has one of the molecular weightsdescribed above (e.g., at least about 3 kDa and less than or equal toabout 15 kDa), and the combined hydrophilic blocks have a weightpercentage with respect to the polymer in one of the ranges describedabove (e.g., at least about 15 wt %, at least about 20 wt %, at leastabout 25 wt %, at least about 30 wt %, at least about 50%, or at leastabout 70% and less than or equal to about 80 wt %). Certain Pluronic®polymers that fall within these criteria include, for example, F127,F108, P105 and P103. Surprisingly, and as described in more detail inthe Examples, it was found that these particular Pluronic® polymersrendered certain particles mucus penetrating more than other Pluronic®polymers tested that did not fall within this criteria. Additionally,other agents that did not render particles mucus penetrating (for somecertain particle cores) included certain polymers such aspolyvinylpyrrolidones (PVP/Kollidon), polyvinyl alcohol-polyethyleneglycol graft-copolymer (Kollicoat IR), hydroxypropyl methylcellulose(Methocel); solutol HS 15, Triton X100, tyloxapol, cremophor RH 40;small molecules such as Span 20, Span 80, octyl glucoside,cetytrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS).

It should be appreciated that the ability of a surface-altering agent torender a particle or core mucus penetrating may depend at least in parton the particular core/surface-altering agent combination, including theability of the surface-altering agent to attach to the core and/or thedensity of the surface-altering agent on the core/particle surface. Assuch, in some embodiments a particular surface-altering agent mayenhance the mobility of one type of particle or core but may not enhancethe mobility of particle or core of another type.

Although much of the description herein may involve coatings comprisinga hydrophilic block-hydrophobic block-hydrophilic block configuration(e.g., a PEG-PPO-PEG triblock copolymer) or coatings comprising asynthetic polymer having pendant hydroxyl groups, it should beappreciated that the coatings are not limited to these configurationsand materials and that other configurations and materials are possible.

Furthermore, although many of the embodiments described herein involve asingle coating, in other embodiments, a particle may include more thanone coating (e.g., at least two, three, four, five, or more coatings),and each coating need not be formed of or comprise a mucus penetratingmaterial. In some cases, an intermediate coating (i.e., a coatingbetween the core surface and an outer coating) may include a polymerthat facilitates attachment of an outer coating to the core surface. Inmany embodiments, an outer coating of a particle includes a polymercomprising a material that facilitates the transport of the particlethrough mucus.

As such, a coating (e.g., an inner coating, an intermediate coating,and/or an outer coating) may include any suitable polymer. In somecases, the polymer may be biocompatible and/or biodegradable. In somecases, the polymeric material may comprise more than one type of polymer(e.g., at least two, three, four, five, or more, polymers). In somecases, a polymer may be a random copolymer or a block copolymer (e.g., adiblock copolymer, a triblock copolymer) as described herein.

Non-limiting examples of suitable polymers may include polyamines,polyethers, polyamides, polyesters, polycarbamates, polyureas,polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes,polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates,polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D, L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane,poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), poly(ethyleneglycol), poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides,polyorthoesters, poly(ester amides), polyamides, poly(ester ethers),polycarbonates, polyalkylenes such as polyethylene and polypropylene,polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkyleneoxides (PEO), polyalkylene terephthalates such as poly(ethyleneterephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinylesters such as poly(vinyl acetate), polyvinyl halides such as poly(vinylchloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS),polyurethanes, derivatized celluloses such as alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers ofacrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointlyreferred to herein as “polyacrylic acids”), and copolymers and mixturesthereof, polydioxanone and its copolymers, polyhydroxyalkanoates,polypropylene fumarate), polyoxymethylene, poloxamers,poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone.

The molecular weight of a polymer may vary. In some embodiments, themolecular weight may be at least about 0.5 kDa, at least about 1 kDa, atleast about 2 kDa, at least about 3 kDa, at least about 4 kDa, at leastabout 5 kDa, at least about 6 kDa, at least about 8 kDa, at least about10 kDa, at least about 12 kDa, at least about 15 kDa, at least about 20kDa, at least about 30 kDa, at least about 40 kDa, or at least about 50kDa. In some embodiments, the molecular weight may be less than or equalto about 50 kDa, less than or equal to about 40 kDa, less than or equalto about 30 kDa, less than or equal to about 20 kDa, less than or equalto about 12 kDa, less than or equal to about 10 kDa, less than or equalto about 8 kDa, less than or equal to about 6 kDa, less than or equal toabout 5 kDa, or less than or equal to about 4 kDa. Combinations of theabove-referenced ranges are possible (e.g., a molecular weight of atleast about 2 kDa and less than or equal to about 15 kDa). Other rangesare also possible. The molecular weight may be determined using anyknown technique such as light-scattering and gel permeationchromatography. Other methods are known in the art.

In certain embodiments, the polymer is biocompatible, i.e., the polymerdoes not typically induce an adverse response when inserted or injectedinto a living subject; for example, it does not include significantinflammation and/or acute rejection of the polymer by the immune system,for instance, via a T-cell-mediated response. It will be recognized, ofcourse, that “biocompatibility” is a relative term, and some degree ofimmune response is to be expected even for polymers that are highlycompatible with living tissue. However, as used herein,“biocompatibility” refers to the acute rejection of material by at leasta portion of the immune system, i.e., a non-biocompatible materialimplanted into a subject provokes an immune response in the subject thatis severe enough such that the rejection of the material by the immunesystem cannot be adequately controlled, and often is of a degree suchthat the material must be removed from the subject. One simple test todetermine biocompatibility is to expose a polymer to cells in vitro;biocompatible polymers are polymers that typically does not result insignificant cell death at moderate concentrations, e.g., atconcentrations of about 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. In some embodiments, asubstance is “biocompatible” if its addition to cells in vitro resultsin less than or equal to 20% cell death, and their administration invivo does not induce unwanted inflammation or other such adverseeffects.

In certain embodiments, a biocompatible polymer may be biodegradable,i.e., the polymer is able to degrade, chemically and/or biologically(e.g., by the cellular machinery or by hydrolysis), within aphysiological environment, such as within the body or when introduced tocells. For instance, the polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), and/orthe polymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer is degraded intomonomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymer maybe biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymer may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (i.e., fewer than about 20% of the cells are killed when thecomponents are added to cells in vitro). For example, polylactide may behydrolyzed to form lactic acid, polyglycolide may be hydrolyzed to formglycolic acid, etc.).

Examples of biodegradable polymers include, but are not limited to,poly(ethylene glycol)-poly(propylene oxide)-poly(ethylene glycol)triblock copolymers, poly(lactide) (or poly(lactic acid)),poly(glycolide) (or poly(glycolic acid)), poly(orthoesters),poly(caprolactones), polylysine, poly(ethylene imine), poly(acrylicacid), poly(urethanes), poly(anhydrides), poly(esters),poly(trimethylene carbonate), poly(ethyleneimine), poly(acrylic acid),poly(urethane), poly(beta amino esters) or the like, and copolymers orderivatives of these and/or other polymers, for example,poly(lactide-co-glycolide) (PLGA).

In certain embodiments, a polymer may biodegrade within a period that isacceptable in the desired application. In certain embodiments, such asin vivo therapy, such degradation occurs in a period usually less thanabout five years, one year, six months, three months, one month, fifteendays, five days, three days, or even one day or less (e.g., 1-4 hours,4-8 hours, 4-24 hours, 1-24 hours) on exposure to a physiologicalsolution with a pH between 6 and 8 having a temperature of between 25and 37° C. In other embodiments, the polymer degrades in a period ofbetween about one hour and several weeks, depending on the desiredapplication.

Although coatings and particles described herein may include polymers,in some embodiments, the particles described herein comprise ahydrophobic material that is not a polymer (e.g., a non-polymer) and isnot a pharmaceutical agent. For example, all or portions of a particlemay be coated with a passivating layer in some embodiments. Non-limitingexamples of non-polymeric materials may include certain metals, waxes,and organic materials (e.g., organic silanes, perfluorinated orfluorinated organic materials).

Particles with Reduced Mucoadhesion

As described herein, in some embodiments, a method involves identifyinga material such as a particle to which it is desired that itsmucoadhesiveness be reduced. Materials in need of increased diffusivitythrough mucus may be, for example, hydrophobic, have many hydrogen bonddonors or acceptors, and/or may be highly charged. In some cases, thematerial may include a crystalline or amorphous solid material. Thematerial, which may serve as a core, may be coated with a suitablepolymer described herein, thereby forming a particle with a plurality ofsurface-altering moieties on the surface, resulting in reducedmucoadhesion. Particles herein described as having reduced mucoadhesionmay alternatively be characterized as having increased transport throughmucus, being mobile in mucus, or mucus-penetrating (i.e.,mucus-penetrating particles), meaning that the particles are transportedthrough mucus faster than a (negative) control particle. The (negative)control particle may be a particle that is known to be mucoadhesive,e.g., an unmodified particle or core that is not coated with a coatingdescribed herein, such as a 200 nm carboxylated polystyrene particle.

In certain embodiments, methods herein include preparing apharmaceutical composition or formulation of the modified substance,e.g., in a formulation adapted for delivery (e.g., topical delivery) tomucus or a mucosal surface of a subject. The pharmaceutical compositionwith surface-altering moieties may be delivered to the mucosal surfaceof a subject, may pass through the mucosal barrier in the subject,and/or prolonged retention and/or increased uniform distribution of theparticles at mucosal surfaces, e.g., due to reduced mucoadhesion. Aswill be known by those of ordinary skill in the art, mucus is aviscoelastic and adhesive substance that traps most foreign particles.Trapped particles are not able to reach the underlying epithelium and/orare quickly eliminated by mucus clearance mechanisms. For a particle toreach the underlying epithelium and/or for a particle to have prolongedretention in the mucosal tissue, the particle must quickly penetratemucus secretions and/or avoid the mucus clearance mechanisms. If aparticle does not adhere substantially to the mucus, the particle may beable to diffuse in the interstitial fluids between mucin fibers andreach the underlying epithelium and/or not be eliminated by the mucusclearance mechanisms. Accordingly, modifying mucoadhesive materials,(e.g., pharmaceutical agents that are hydrophobic) with a material toreduce the mucoadhesion of the particle may allow for efficient deliveryof the particles to the underlying epithelium and/or prolonged retentionat mucosal surfaces.

Furthermore, in some embodiments, the particles described herein havingreduced mucoadhesion facilitate better distribution of the particles ata tissue surface, and/or have a prolonged presence at the tissuesurface, compared to particles that are more mucoadhesive. For example,in some cases a luminal space such as the gastrointestinal tract issurrounded by a mucus-coated surface. Mucoadhesive particles deliveredto such a space are typically removed from the luminal space and fromthe mucus-coated surface by the body's natural clearance mechanisms. Theparticles described herein with reduced mucoadhesion may remain in theluminal space for relatively longer periods compared to the mucoadhesiveparticles. This prolonged presence may prevent or reduce clearance ofthe particles, and/or may allow for better distribution of the particleson the tissue surface. The prolonged presence may also affect theparticle transport through the luminal space, e.g., the particles maydistribute into the mucus layer and may reach the underlying epithelium.

In certain embodiments, a material (e.g., a core) coated with a polymerdescribed herein may pass through mucus or a mucosal barrier in asubject, and/or exhibit prolonged retention and/or increase uniformdistribution of the particles at mucosal surfaces, e.g., such substancesare cleared more slowly (e.g., at least 2 times, 5 times, 10 times, oreven at least 20 times more slowly) from a subject's body as compared toa (negative) control particle. The (negative) control particle may be aparticle that is known to be mucoadhesive, e.g., an unmodified particleor core that is not coated with a coating described herein, such as a200 nm carboxylated polystyrene particle.

In certain embodiments, a particle described herein has certain arelative velocity, <V_(mean)>_(rel), which is defined as follows:

$\begin{matrix}{{\langle V_{mean}\rangle}_{rel} = \frac{{\langle V_{mean}\rangle}_{Sample} - {\langle V_{mean}\rangle}_{Neg{ative}\mspace{14mu} {control}}}{{\langle V_{mean}\rangle}_{Pos{itive}\mspace{14mu} {control}} - {\langle V_{mean}\rangle}_{Neg{ative}\mspace{14mu} {control}}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

where <V_(mean)> is the ensemble average trajectory-mean velocity,V_(mean) is the velocity of an individual particle averaged over itstrajectory, the sample is the particle of interest, the negative controlis a 200 nm carboxylated polystyrene particle, and the positive controlis a 200 nm polystyrene particle densely PEGylated with 2 kDa-5 kDa PEG.

The relative velocity can be measured by a multiple particle trackingtechnique. For instance, a fluorescent microscope equipped with a CCDcamera can be used to capture 15 s movies at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample, negative control, andpositive control. The sample, negative and positive controls may befluorescent particles to observe tracking. Alternatively non-fluorescentparticles may be coated with a fluorescent molecule, a fluorescentlytagged surface agent or a fluorescently tagged polymer. An advancedimage processing software (e.g., Image Pro or MetaMorph) can be used tomeasure individual trajectories of multiple particles over a time-scaleof at least 3.335 s (50 frames).

In some embodiments, a particle described herein has a relative velocityof greater than or equal to about 0.3, greater than or equal to about0.4, greater than or equal to about 0.5, greater than or equal to about0.6, greater than or equal to about 0.7, greater than or equal to about0.8, greater than or equal to about 0.9, greater than or equal to about1.0, greater than or equal to about 1.1, greater than or equal to about1.2, greater than or equal to about 1.3, greater than or equal to about1.4, greater than or equal to about 1.5, greater than or equal to about1.6, greater than or equal to about 1.7, greater than or equal to about1.8, greater than or equal to about 1.9 or greater than or equal toabout 2.0 in mucus. In some embodiments, a particle described herein hasa relative velocity of less than or equal to about 10.0, less than orequal to about 8.0, less than or equal to about 6.0, less than or equalto about 4.0, less than or equal to about 3.0, less than or equal toabout 2.0, less than or equal to about 1.9, less than or equal to about1.8, less than or equal to about 1.7, less than or equal to about 1.6,less than or equal to about 1.5, less than or equal to about 1.4, lessthan or equal to about 1.3, less than or equal to about 1.2, less thanor equal to about 1.1, less than or equal to about 1.0, less than orequal to about 0.9, less than or equal to about 0.8, or less than orequal to about 1.7 in mucus. Combinations of the above-noted ranges arepossible (e.g., a relative velocity of greater than or equal to about0.5 and less than or equal to about 6.0). Other ranges are alsopossible. The mucus may be, for example, human cervicovaginal mucus.

In certain embodiments, a particle described herein can diffuse throughmucus or a mucosal barrier at a greater rate or diffusivity than acontrol particle or a corresponding particle (e.g., a correspondingparticle that is unmodified and/or is not coated with a coatingdescribed herein). In some cases, a particle described herein may passthrough mucus or a mucosal barrier at a rate of diffusivity that is atleast about 10 times, 20 times, 30 times, 50 times, 100 times, 200times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, ormore, higher than a control particle or a corresponding particle. Insome cases, a particle described herein may pass through mucus or amucosal barrier at a rate of diffusivity that is less than or equal toabout 10000 times higher, less than or equal to about 5000 times higher,less than or equal to about 2000 times higher, less than or equal toabout 1000 times higher, less than or equal to about 500 times higher,less than or equal to about 200 times higher, less than or equal toabout 100 times higher, less than or equal to about 50 times higher,less than or equal to about 30 times higher, less than or equal to about20 times higher, or less than or equal to about 10 times higher than acontrol particle or a corresponding particle. Combinations of theabove-referenced ranges are also possible (e.g., at least about 10 timesand less than or equal to about 1000 times higher than a controlparticle or a corresponding particle). Other ranges are also possible.

For the purposes of the comparisons described herein, the correspondingparticle may be approximately the same size, shape, and/or density asthe test particle but lacking the coating that makes the test particlemobile in mucus. In some cases, the measurement is based on a time scaleof about 1 second, or about 0.5 second, or about 2 seconds, or about 5seconds, or about 10 seconds. Those of ordinary skill in the art will beaware of methods for determining the geometric mean square displacementand rate of diffusivity.

In addition, a particle described herein may pass through mucus or amucosal barrier with a geometric mean squared displacement that is atleast about 10 times, 20 times, 30 times, 50 times, 100 times, 200times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, ormore, higher than a corresponding particle or control particle. In somecases, a particle described herein may pass through mucus or a mucosalbarrier with a geometric mean squared displacement that is less than orequal to about 10000 times higher, less than or equal to about 5000times higher, less than or equal to about 2000 times higher, less thanor equal to about 1000 times higher, less than or equal to about 500times higher, less than or equal to about 200 times higher, less than orequal to about 100 times higher, less than or equal to about 50 timeshigher, less than or equal to about 30 times higher, less than or equalto about 20 times higher, or less than or equal to about 10 times higherthan a control particle or a corresponding particle. Combinations of theabove-referenced ranges are also possible (e.g., at least about 10 timesand less than or equal to about 1000 times higher than a controlparticle or a corresponding particle). Other ranges are also possible.

In some embodiments, a particle described herein diffuses through amucosal barrier at a rate approaching the rate or diffusivity at whichsaid particles can diffuse through water. In some cases, a particledescribed herein may pass through a mucosal barrier at a rate ordiffusivity that is less than or equal to about 1/100, less than orequal to about 1/200, less than or equal to about 1/300, less than orequal to about 1/400, less than or equal to about 1/500, less than orequal to about 1/600, less than or equal to about 1/700, less than orequal to about 1/800, less than or equal to about 1/900, less than orequal to about 1/1000, less than or equal to about 1/2000, less than orequal to about 1/5000, less than or equal to about 1/10,000 thediffusivity that the particle diffuse through water under identicalconditions. In some cases, a particle described herein may pass througha mucosal barrier at a rate or diffusivity that is greater than or equalto about 1/10,000, greater than or equal to about 1/5000, greater thanor equal to about 1/2000, greater than or equal to about 1/1000, greaterthan or equal to about 1/900, greater than or equal to about 1/800,greater than or equal to about 1/700, greater than or equal to about1/600, greater than or equal to about 1/500, greater than or equal toabout 1/400, greater than or equal to about 1/300, greater than or equalto about 1/200, or greater than or equal to about 1/100 the diffusivitythat the particle diffuse through water under identical conditions.Combinations of the above-referenced ranges are also possible (e.g.,greater than or equal to about 1/5000 and less than 1/500 thediffusivity that the particle diffuse through water under identicalconditions). Other ranges are also possible. The measurement may bebased on a time scale of about 1 second, or about 0.5 second, or about 2seconds, or about 5 seconds, or about 10 seconds.

In a particular embodiment, a particle described herein may diffusethrough human cervicovaginal mucus at a diffusivity that is less thanabout 1/500 the diffusivity that the particle diffuses through water. Insome cases, the measurement is based on a time scale of about 1 second,or about 0.5 second, or about 2 seconds, or about 5 seconds, or about 10seconds.

In certain embodiments, the present invention provides particles thattravel through mucus, such as human cervicovaginal mucus, at certainabsolute diffusivities. For example, the particles of described hereinmay travel at diffusivities of at least about 1×10⁻⁴ μm/s, 2×10⁻⁴ μm/s,5×10⁻⁴ μm/s, 1×10⁻³ μm/s, 2×10⁻³ μm/s, 5×10⁻³ μm/s, 1×10⁻² μm/s, 2×10⁻²μm/s, 4×10⁻² μm/s, 5×10⁻² μm/s, 6×10⁻² μm/s, 8×10⁻² μm/s, 1×10⁻¹ μm/s,2×10⁻¹ μm/s, 5×10⁻¹ μm/s, 1 μm/s, or 2 μm/s. In some cases, theparticles may travel at diffusivities of less than or equal to about 2μm/s, less than or equal to about 1 μm/s, less than or equal to about5×10⁻¹ μm/s, less than or equal to about 2×10⁻¹ μm/s, less than or equalto about 1×10⁻¹ μm/s, less than or equal to about 8×10⁻² μm/s, less thanor equal to about 6×10⁻² μm/s, less than or equal to about 5×10⁻² μm/s,less than or equal to about 4×10⁻² μm/s, less than or equal to about2×10⁻² μm/s, less than or equal to about 1×10⁻² μm/s, less than or equalto about 5×10⁻³ μm/s, less than or equal to about 2×10⁻³ μm/s, less thanor equal to about 1×10⁻³ μm/s, less than or equal to about 5×10⁻⁴ μm/s,less than or equal to about 2×10⁻⁴ μm/s, or less than or equal to about1×10⁻⁴ μm/s. Combinations of the above-referenced ranges are alsopossible (e.g., greater than or equal to about 2×10⁻⁴ μm/s and less thanor equal to about 1×10⁻¹ μm/s). Other ranges are also possible. In somecases, the measurement is based on a time scale of about 1 second, orabout 0.5 second, or about 2 seconds, or about 5 seconds, or about 10seconds.

It should be appreciated that while many of the mobilities (e.g.,relative velocities, diffusivities) described here may be measured inhuman cervicovaginal mucus, they may be measured in other types of mucusas well.

In certain embodiments, a particle described herein comprisessurface-altering moieties at a given density. The surface-alteringmoieties may be the portions of a surface-altering agent that are, forexample, exposed to the solvent containing the particle. As an example,the hydrolyzed units/blocks of PVA may be surface-altering moieties ofthe surface-altering agent PVA. In another example, the PEG segments maybe surface-altering moieties of the surface-altering agent PEG-PPO-PEG.In some cases, the surface-altering moieties and/or surface-alteringagents are present at a density of at least about 0.001 units ormolecules per nm², at least about 0.002, at least about 0.005, at leastabout 0.01, at least about 0.02, at least about 0.05, at least about0.1, at least about 0.2, at least about 0.5, at least about 1, at leastabout 2, at least about 5, at least about 10, at least about 20, atleast about 50, at least about 100 units or molecules per nm², or moreunits or molecules per nm². In some cases, the surface-altering moietiesand/or surface-altering agents are present at a density of less than orequal to about 100 units or molecules per nm², less than or equal toabout 50, less than or equal to about 20, less than or equal to about10, less than or equal to about 5, less than or equal to about 2, lessthan or equal to about 1, less than or equal to about 0.5, less than orequal to about 0.2, less than or equal to about 0.1, less than or equalto about 0.05, less than or equal to about 0.02, or less than or equalto about 0.01 units or molecules per nm². Combinations of theabove-referenced ranges are possible (e.g., a density of at least about0.01 and less than or equal to about 1 units or molecules per nm²).Other ranges are also possible. In some embodiments, the density valuesdescribed above may be an average density as the surface altering agentis in equilibrium with other components in solution.

Those of ordinary skill in the art will be aware of methods to estimatethe average density of surface-altering moieties (see, for example, S.J. Budijono et al., Colloids and Surfaces A: Physicochem. Eng. Aspects360 (2010) 105-110 and Joshi, et al., Anal. Chim. Acta 104 (1979)153-160, each of which is incorporated herein by reference). Forexample, as described herein, the average density of surface-alteringmoieties can be determined using HPLC quantitation and DLS analysis. Asuspension of particles for which surface density determination is ofinterest is first sized using DLS: a small volume is diluted to anappropriate concentration (˜100 μg/mL, for example), and the z-averagediameter is taken as a representative measurement of particle size. Theremaining suspension is then divided into two aliquots. Using HPLC, thefirst aliquot is assayed for the total concentration of core materialand for the total concentration of surface-altering moiety. Again usingHPLC the second aliquot is assayed for the concentration of free orunbound surface-altering moiety. In order to get only the free orunbound surface-altering moiety from the second aliquot, the particles,and therefore any bound surface-altering moiety, are removed byultracentrifugation. By subtracting the concentration of the unboundsurface-altering moiety from the total concentration of surface-alteringmoiety, the concentration of bound surface-altering moiety can bedetermined. Since the total concentration of core material was alsodetermined from the first aliquot, the mass ratio between the corematerial and the surface-altering moiety can be determined. Using themolecular weight of the surface-altering moiety the number ofsurface-altering moiety to mass of core material can be calculated. Toturn this number into a surface density measurement, the surface areaper mass of core material needs to be calculated. The volume of theparticle is approximated as that of a sphere with the diameter obtainedfrom DLS allowing for the calculation of the surface area per mass ofcore material. In this way the number of surface-altering moieties persurface area can be determined.

In certain embodiments, the particles described herein comprisesurface-altering moieties and/or agents that affect the zeta-potentialof the particle. The zeta potential of the coated particle may be, forexample, at least about −100 mV, at least about −75 mV, at least about−50 mV, at least about −40 mV, at least about −30 mV, at least about −20mV, at least about −10 mV, at least about −5 mV, at least about 5 mV, atleast about 10 mV, at least about 20 mV, at least about 30 mV, at leastabout 40 mV, at least about 50 mV, at least about 75 mV, or at leastabout 100 mV. Combinations of the above-referenced ranges are possible(e.g., a zeta-potential of at least about −50 mV and less than or equalto about 50 mV). Other ranges are also possible.

The coated particles described herein may have any suitable shape and/orsize. In some embodiments, a coated particle has a shape substantiallysimilar to the shape of the core. In some cases, a coated particledescribed herein may be a nanoparticle, i.e., the particle has acharacteristic dimension of less than about 1 micrometer, where thecharacteristic dimension of the particle is the diameter of a perfectsphere having the same volume as the particle. In other embodiments,larger sizes are possible (e.g., about 1-10 microns). A plurality ofparticles, in some embodiments, may also be characterized by an averagesize (e.g., an average largest cross-sectional dimension, or an averagesmallest cross-sectional dimension for the plurality of particles). Aplurality of particles may have an average size of, for example, lessthan or equal to about 10 μm, less than or equal to about 5 μm, lessthan or equal to about 1 μm, less than or equal to about 800 nm, lessthan or equal to about 700 nm, less than or equal to about 500 nm, lessthan or equal to 400 nm, less than or equal to 300 nm, less than orequal to about 200 nm, less than or equal to about 100 nm, less than orequal to about 75 nm, less than or equal to about 50 nm, less than orequal to about 40 nm, less than or equal to about 35 nm, less than orequal to about 30 nm, less than or equal to about 25 nm, less than orequal to about 20 nm, less than or equal to about 15 nm, or less than orequal to about 5 nm. In some cases, a plurality of particles may have anaverage size of, for example, at least about 5 nm, at least about 20 nm,at least about 50 nm, at least about 100 nm, at least about 200 nm, atleast about 300 nm, at least about 400 nm, at least about 500 nm, atleast about 1 μm, at least or at least about 5 μm. Combinations of theabove-referenced ranges are also possible (e.g., an average size of atleast about 50 nm and less than or equal to about 500 nm). In oneembodiment, a plurality of particles have an average size between about100 nm and 500 nm, 200 nm and 500 nm, 200 nm and 400 nm, or 200 nm and300 nm. Other ranges are also possible. In some embodiments, the sizesof the cores formed by a process described herein have a Gaussian-typedistribution.

Pharmaceutical Agents

In some embodiments, a coated particle comprises at least onepharmaceutical agent. The pharmaceutical agent may be present in thecore of the particle and/or present in a coating of the particle (e.g.,dispersed throughout the core and/or coating). In some cases, apharmaceutical agent may be disposed on the surface of the particle(e.g., on an outer surface of a coating, the inner surface of a coating,on a surface of the core). The pharmaceutical agent may be containedwithin a particle and/or disposed in a portion of the particle usingcommonly known techniques (e.g., by coating, adsorption, covalentlinkage, encapsulation, or other process). In some cases, thepharmaceutical agent may be present in the core of the particle prior toor during coating of the particle. In some cases, the pharmaceuticalagent is present during the formation of the core of the particle, asdescribed herein.

Non-limiting examples of pharmaceutical agents include imaging agents,diagnostic agents, therapeutic agents, agents with a detectable label,nucleic acids, nucleic acid analogs, small molecules, peptidomimetics,proteins, peptides, lipids, vaccines, viral vectors, virus, andsurfactants.

In some embodiments, a pharmaceutical agent contained in a particledescribed herein has a therapeutic, diagnostic, or imaging effect in amucosal tissue to be targeted. Non-limiting examples of mucosal tissuesinclude oral (e.g., including the buccal and esophagal membranes andtonsil surface), ophthalmic, gastrointestinal (e.g., including stomach,small intestine, large intestine, colon, rectum), nasal, respiratory(e.g., including nasal, pharyngeal, tracheal and bronchial membranes),and genital (e.g., including vaginal, cervical and urethral membranes)tissues.

Any suitable number of pharmaceutical agents may be present in aparticle described herein. For example, at least 1, at least 2, at least3, at least 4, at least 5, or more, but generally less than 10,pharmaceutical agents may be present in a particle described herein.

A number of drugs that are mucoadhesive are known in the art and may beused as pharmaceutical agents in the particles described herein (see,for example, Khanvilkar K, Donovan M D, Flanagan D R, Drug transferthrough mucus, Advanced Drug Delivery Reviews 48 (2001) 173-193; Bhat PG, Flanagan D R, Donovan M D. Drug diffusion through cystic fibroticmucus: steady-state permeation, rheologic properties, and glycoproteinmorphology, J Pharm Sci, 1996 Jun.; 85(6):624-30). Additionalnon-limiting examples of pharmaceutical agents include imaging anddiagnostic agents (such as radioopaque agents, labeled antibodies,labeled nucleic acid probes, dyes, such as colored or fluorescent dyes,etc.) and adjuvants (radiosensitizers, transfection-enhancing agents,chemotactic agents and chemoattractants, peptides that modulate celladhesion and/or cell mobility, cell permeabilizing agents, vaccinepotentiators, inhibitors of multidrug resistance and/or efflux pumps,etc.).

Additional non-limiting examples of pharmaceutical agents includepazopanib, sorafenib, lapatinib, fluocinolone acetonide, semaxanib,axitinib, tivozanib, cediranib, linifanib, regorafenib, telatinib,vatalanib, MGCD-265, OSI-930, KRN-633, bimatoprost, latanoprost,travoprost, aloxiprin, auranofin, azapropazone, benorylate, diflunisal,etodolac, fenbufen, fenoprofen calcim, flurbiprofen, furosemide,ibuprofen, indomethacin, ketoprofen, loteprednol etabonate, bromfenacberyllium, bromfenac magnesium, bromfenac calcium, bromfenac strontium,bromfenac barium, bromfenac zinc, bromfenac copper(II), diclofenac freeacid, diclofenac beryllium, diclofenac magnesium, diclofenac calcium,diclofenac strontium, diclofenac barium, diclofenac zinc, diclofenaccopper(II), ketorolac free acid, ketorolac beryllium, ketorolacmagnesium, ketorolac calcium, ketorolac strontium, ketorolac barium,ketorolac zinc, ketorolac copper(II), meclofenamic acid, mefenamic acid,nabumetone, naproxen, oxyphenbutazone, phenylbutazone, piroxicam,sulindac, albendazole, bephenium hydroxynaphthoate, cambendazole,dichlorophen, ivermectin, mebendazole, oxamniquine, oxfendazole, oxantelembonate, praziquantel, pyrantel embonate, thiabendazole, amiodaroneHCl, disopyramide, flecainide acetate, quinidine sulphate.Anti-bacterial agents: benethamine penicillin, cinoxacin, ciprofloxacinHCl, clarithromycin, clofazimine, cloxacillin, demeclocycline,doxycycline, erythromycin, ethionamide, imipenem, nalidixic acid,nitrofurantoin, rifampicin, spiramycin, sulphabenzamide, sulphadoxine,sulphamerazine, sulphacetamide, sulphadiazine, sulphafurazole,sulphamethoxazole, sulphapyridine, tetracycline, trimethoprim,dicoumarol, dipyridamole, nicoumalone, phenindione, amoxapine,maprotiline HCl, mianserin HCL, nortriptyline HCl, trazodone HCL,trimipramine maleate, acetohexamide, chlorpropamide, glibenclamide,gliclazide, glipizide, tolazamide, tolbutamide, beclamide,carbamazepine, clonazepam, ethotoin, methoin, methsuximide,methylphenobarbitone, oxcarbazepine, paramethadione, phenacemide,phenobarbitone, phenytoin, phensuximide, primidone, sulthiame, valproicacid, amphotericin, butoconazole nitrate, clotrimazole, econazolenitrate, fluconazole, flucytosine, griseofulvin, itraconazole,ketoconazole, miconazole, natamycin, nystatin, sulconazole nitrate,terbinafine HCl, terconazole, tioconazole, undecenoic acid, allopurinol,probenecid, sulphin-pyrazone, amlodipine, benidipine, darodipine,dilitazem HCl, diazoxide, felodipine, guanabenz acetate, isradipine,minoxidil, nicardipine HCl, nifedipine, nimodipine, phenoxybenzamineHCl, prazosin HCL, reserpine, terazosin HCL, amodiaquine, chloroquine,chlorproguanil HCl, halofantrine HCl, mefloquine HCl, roguanil HCl,pyrimethamine, quinine sulphate, dihydroergotamine mesylate, ergotaminetartrate, methysergide maleate, pizotifen maleate, sumatriptansuccinate, atropine, benzhexol HCl, biperiden, ethopropazine HCl,hyoscyamine, mepenzolate bromide, oxyphencylcimine HCl, tropicamide,aminoglutethimide, amsacrine, azathioprine, busulphan, chlorambucil,cyclosporin, dacarbazine, estramustine, etoposide, lomustine, melphalan,mercaptopurine, methotrexate, mitomycin, mitotane, mitozantrone,procarbazine HCl, tamoxifen citrate, testolactone, benznidazole,clioquinol, decoquinate, diiodohydroxyquinoline, diloxanide furoate,dinitolmide, furzolidone, metronidazole, nimorazole, nitrofurazone,ornidazole, tinidazole, carbimazole, propylthiouracil, alprazolam,amylobarbitone, barbitone, bentazepam, bromazepam, bromperidol,brotizolam, butobarbitone, carbromal, chlordiazepoxide, chlormethiazole,chlorpromazine, clobazam, clotiazepam, clozapine, diazepam, droperidol,ethinamate, flunanisone, flunitrazepam, fluopromazine, flupenthixoldecanoate, fluphenazine decanoate, flurazepam, haloperidol, lorazepam,lormetazepam, medazepam, meprobamate, methaqualone, midazolam,nitrazepam, oxazepam, pentobarbitone, perphenazine pimozide,prochlorperazine, sulpiride, temazepam, thioridazine, triazolam,zopiclone, acebutolol, alprenolol, atenolol, labetalol, metoprolol,nadolol, oxprenolol, pindolol, propranolol, amrinone, digitoxin,digoxin, enoximone, lanatoside C, medigoxin, beclomethasone,betamethasone, budesonide, cortisone acetate, desoxymethasone,dexamethasone, fludrocortisone acetate, flunisolide, flucortolone,fluticasone propionate, hydrocortisone, methylprednisolone,prednisolone, prednisone, triamcinolone, acetazolamide, amiloride,bendrofluazide, bumetanide, chlorothiazide, chlorthalidone, ethacrynicacid, frusemide, metolazone, spironolactone, triamterene, bromocriptinemesylate, lysuride maleate, bisacodyl, cimetidine, cisapride,diphenoxylate HCl, domperidone, famotidine, loperamide, mesalazine,nizatidine, omeprazole, ondansetron HCL, ranitidine HCl, sulphasalazine,acrivastine, astemizole, cinnarizine, cyclizine, cyproheptadie HCl,dimenhydrinate, flunarizine HCl, loratadine, meclozine HCl, oxatomide,terfenadine, bezafibrate, clofibrate, fenofibrate, gemfibrozil,probucol, amyl nitrate, glyceryl trinitrate, isosorbide dinitrate,isosorbide mononitrate, pentaerythritol tetranitrate, betacarotene,vitamin A, vitamin B 2, vitamin D, vitamin E, vitamin K, codeine,dextropropyoxyphene, diamorphine, dihydrocodeine, meptazinol, methadone,morphine, nalbuphine, pentazocine, clomiphene citrate, danazol, ethinylestradiol, medroxyprogesterone acetate, mestranol, methyltestosterone,norethisterone, norgestrel, estradiol, conjugated oestrogens,progesterone, stanozolol, stibestrol, testosterone, tibolone,amphetamine, dexamphetamine, dexfenfluramine, fenfluramine, andmazindol.

Uses and Pharmaceutical Compositions

The particles described herein may be employed in any suitableapplication. In some cases, the particles are part of pharmaceuticalcompositions (e.g., as described herein), for example, those used todeliver a pharmaceutical agent (e.g., a drug, therapeutic agent,diagnostic agent, imaging agent) through or to mucus or a mucosalsurface. A pharmaceutical composition may comprise at least one particledescribed herein and one or more pharmaceutically acceptable excipientsor carriers. The composition may be used in treating, preventing, and/ordiagnosing a condition in a subject, wherein the method comprisesadministering to a subject the pharmaceutical composition. A subject orpatient to be treated by the articles and methods described herein maymean either a human or non-human animal, such as primates, mammals, andvertebrates.

Methods involving treating a subject may include preventing a disease,disorder or condition from occurring in the subject which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it; inhibiting the disease, disorder orcondition, e.g., impeding its progress; and relieving the disease,disorder, or condition, e.g., causing regression of the disease,disorder and/or condition. Treating the disease or condition includesameliorating at least one symptom of the particular disease orcondition, even if the underlying pathophysiology is not affected (e.g.,such treating the pain of a subject by administration of an analgesicagent even though such agent does not treat the cause of the pain).

In some embodiments, a pharmaceutical composition described herein isdelivered to a mucosal surface in a subject and may pass through amucosal barrier in the subject (e.g., mucus), and/or may exhibitprolonged retention and/or increased uniform distribution of theparticles at mucosal surfaces, e.g., due to reduced mucoadhesion.Non-limiting examples of mucosal tissues include oral (e.g., includingthe buccal and esophagal membranes and tonsil surface), ophthalmic,gastrointestinal (e.g., including stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., including nasal,pharyngeal, tracheal and bronchial membranes), genital (e.g., includingvaginal, cervical and urethral membranes).

Pharmaceutical compositions described herein and for use in accordancewith the articles and methods described herein may include apharmaceutically acceptable excipient or carrier. A pharmaceuticallyacceptable excipient or pharmaceutically acceptable carrier may includea non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any suitable type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose, and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator. As would be appreciated by one of skill in this art, theexcipients may be chosen based on the route of administration asdescribed below, the pharmaceutical agent being delivered, time courseof delivery of the agent, etc.

Pharmaceutical compositions containing the particles described hereinmay be administered to a subject via any route known in the art. Theseinclude, but are not limited to, oral, sublingual, nasal, intradermal,subcutaneous, intramuscular, rectal, vaginal, intravenous,intraarterial, intracisternally, intraperitoneal, intravitreal,periocular, topical (as by powders, creams, ointments, or drops), buccaland inhalational administration. In some embodiments, compositionsdescribed herein may be administered parenterally as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. As would be appreciated by one of skillin this art, the route of administration and the effective dosage toachieve the desired biological effect may be determined by the agentbeing administered, the target organ, the preparation beingadministered, time course of administration, disease being treated,intended use, etc.

As an example, the particles may be included in a pharmaceuticalcomposition to be formulated as a nasal spray, such that thepharmaceutical composition is delivered across a nasal mucus layer. Asanother example, the particles may be included in a pharmaceuticalcomposition to be formulated as an inhaler, such that the pharmaceuticalcompositions is delivered across a pulmonary mucus layer. As anotherexample, if compositions are to be administered orally, it may beformulated as tablets, capsules, granules, powders, or syrups.Similarly, the particles may be included in a pharmaceutical compositionthat is to be delivered via ophthalmic, gastrointestinal, nasal,respiratory, rectal, urethral and/or vaginal tissues.

For application by the ophthalmic mucous membrane route, subjectcompositions may be formulated as eye drops or eye ointments. Theseformulations may be prepared by conventional means, and, if desired, thesubject compositions may be mixed with any conventional additive, suchas a buffering or pH-adjusting agents, tonicity adjusting agents,viscosity modifiers, suspension stabilizers, preservatives, and otherpharmaceutical excipients. In addition, in certain embodiments, subjectcompositions described herein may be lyophilized or subjected to anotherappropriate drying technique such as spray drying.

In some embodiments, particles described herein that may be administeredin inhalant or aerosol formulations comprise one or more pharmaceuticalagents, such as adjuvants, diagnostic agents, imaging agents, ortherapeutic agents useful in inhalation therapy. The particle size ofthe particulate medicament should be such as to permit inhalation ofsubstantially all of the medicament into the lungs upon administrationof the aerosol formulation and may be, for example, less than about 20microns, e.g., in the range of about 1 to about 10 microns, e.g., about1 to about 5 microns, although other ranges are also possible. Theparticle size of the medicament may be reduced by conventional means,for example by milling or micronisation. Alternatively, the particulatemedicament can be administered to the lungs via nebulization of asuspension. The final aerosol formulation may contain, for example,between 0.005-90% w/w, between 0.005-50%, between 0.005-10%, betweenabout 0.005-5% w/w, or between 0.01-1.0% w/w, of medicament relative tothe total weight of the formulation. Other ranges are also possible.

It is desirable, but by no means required, that the formulationsdescribed herein contain no components which may provoke the degradationof stratospheric ozone. In particular, in some embodiments, propellantsare selected that do not contain or do not consist essentially ofchlorofluorocarbons such as CCl₃F, CCl₂F₂, and CF₃CCl₃.

The aerosol may comprise propellant. The propellant may optionallycontain an adjuvant having a higher polarity and/or a higher boilingpoint than the propellant. Polar adjuvants which may be used include(e.g., C₂₋₆ aliphatic alcohols and polyols such as ethanol, isopropanol,and propylene glycol, preferably ethanol. In general, only smallquantities of polar adjuvants (e.g., 0.05-3.0% w/w) may be required toimprove the stability of the dispersion—the use of quantities in excessof 5% w/w may tend to dissolve the medicament. Formulations inaccordance with the embodiments described herein may contain less than1% w/w, e.g., about 0.1% w/w, of polar adjuvant. However, theformulations described herein may be substantially free of polaradjuvants, especially ethanol. Suitable volatile adjuvants includesaturated hydrocarbons such as propane, n-butane, isobutane, pentane andisopentane and alkyl ethers such as dimethyl ether. In general, up to50% w/w of the propellant may comprise a volatile adjuvant, for example,up to 30% w/w of a volatile saturated C₁-C₆ hydrocarbon. Optionally, theaerosol formulations according to the invention may further comprise oneor more surfactants. The surfactants can be physiologically acceptableupon administration by inhalation. Within this category are includedsurfactants such as L-α-phosphatidylcholine (PC),1,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitantrioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylenesorbitan monolaurate, polyoxyethylene sorbitan monooleate, naturallecithin, oleyl polyoxyethylene ether, stearyl polyoxyethylene ether,lauryl polyoxyethylene ether, block copolymers of oxyethylene andoxypropylene, synthetic lecithin, diethylene glycol dioleate,tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glycerylmonooleate, glyceryl monostearate, glyceryl monoricinoleate, cetylalcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridiniumchloride, benzalkonium chloride, olive oil, glyceryl monolaurate, cornoil, cotton seed oil, and sunflower seed oil.

The formulations described herein may be prepared by dispersal of theparticles in the selected propellant and/or co-propellant in anappropriate container, e.g., with the aid of sonication. The particlesmay be suspended in co-propellant and filled into a suitable container.The valve of the container is then sealed into place and the propellantintroduced by pressure filling through the valve in the conventionalmanner. The particles may be thus suspended or dissolved in a liquifiedpropellant, sealed in a container with a metering valve and fitted intoan actuator. Such metered dose inhalers are well known in the art. Themetering valve may meter 10 to 500 μL and preferably 25 to 150 μL. Incertain embodiments, dispersal may be achieved using dry powder inhalers(e.g., spinhaler) for the particles (which remain as dry powders). Inother embodiments, nanospheres, may be suspended in an aqueous fluid andnebulized into fine droplets to be aerosolized into the lungs.

Sonic nebulizers may be used because they minimize exposing the agent toshear, which may result in degradation of the particles. Ordinarily, anaqueous aerosol is made by formulating an aqueous solution or suspensionof the particles together with conventional pharmaceutically acceptablecarriers and stabilizers. The carriers and stabilizers vary with therequirements of the particular composition, but typically includenon-ionic surfactants (Tweens, Pluronic®, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars, or sugaralcohols. Aerosols generally are prepared from isotonic solutions.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, liposomes, micelles, polynucleotide/lipidcomplexes), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Incertain embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration can be suppositorieswhich can be prepared by mixing the particles with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol, or a suppository wax which are solid at ambient temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the particles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles described herein, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles describedherein, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

The particles described herein comprising a pharmaceutical agent may beadministered to a subject to be delivered in an amount sufficient todeliver to a subject a therapeutically effective amount of anincorporated pharmaceutical agent as part of a diagnostic, prophylactic,or therapeutic treatment. In general, an effective amount of apharmaceutical agent or component refers to the amount necessary toelicit the desired biological response. The desired concentration ofpharmaceutical agent in the particle will depend on numerous factors,including, but not limited to, absorption, inactivation, and excretionrates of the drug as well as the delivery rate of the compound from thesubject compositions, the desired biological endpoint, the agent to bedelivered, the target tissue, etc. It is to be noted that dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions. Typically, dosingwill be determined using techniques known to one skilled in the art.

The concentration and/or amount of any pharmaceutical agent to beadministered to a subject may be readily determined by one of ordinaryskill in the art. Known methods are also available to assay local tissueconcentrations, diffusion rates from particles and local blood flowbefore and after administration of the therapeutic formulation.

The compositions and/or formulations described herein may have anysuitable osmolarity. In some embodiments, a composition and/orformulation described herein may have an osmolarity of at least about 0mOsm/L, at least about 5 mOsm/L, at least about 25 mOsm/L, at leastabout 50 mOsm/L, at least about 75 mOsm/L, at least about 100 mOsm/L, atleast about 150 mOsm/L, at least about 200 mOsm/L, at least about 250mOsm/L, or at least about 310 mOsm/L. In certain embodiments, acomposition and/or formulation described herein may have an osmolarityof less than or equal to about 310 mOsm/L, less than or equal to about250 mOsm/L, less than or equal to about 200 mOsm/L, less than or equalto about 150 mOsm/L, less than or equal to about 100 mOsm/L, less thanor equal to about 75 mOsm/L, less than or equal to about 50 mOsm/L, lessthan or equal to about 25 mOsm/L, or less than or equal to about 5mOsm/L. Combinations of the above-referenced ranges are also possible(e.g., an osmolarity of at least about 0 mOsm/L and less than or equalto about 50 mOsm/L). Other ranges are also possible. The osmolarity ofthe composition and/or formulation can be varied by changing, forexample, the concentration of salts present in the solvent of thecomposition and/or formulation.

In one set of embodiments, a composition and/or formulation includes acore material comprises a drug, such as loteprednol etabonate,sorafenib, linifanib, MGCD-265, pazopanib, cediranib, axitinib,bromfenac calcium, diclofenac (e.g., diclofenac free acid or a divalentor trivalent metal salt thereof), ketorolac (e.g., ketorolac free acidor a divalent or trivalent metal salt thereof), or other suitable drugdescribed herein. In some embodiments, the ratio of the weight of thedrug to the weight of the one or more surface-altering agents (e.g.,Pluronic® F127) present in the composition and/or formulation is greaterthan or equal to about 1:100, greater than or equal to about 1:30,greater than or equal to about 1:10, greater than or equal to about 1:3,greater than or equal to about 1:1, greater than or equal to about 3:1,greater than or equal to about 10:1, greater than or equal to about30:1, or greater than or equal to about 100:1. In some embodiments, theratio of the weight of the drug to the weight of the one or moresurface-altering agents in a composition and/or formulation is less thanor equal to about 100:1, less than or equal to about 30:1, less than orequal to about 10:1, less than or equal to about 3:1, less than or equalto about 1:1, less than or equal to about 1:3: less than or equal toabout 1:10, less than or equal to about 1:30, or less than or equal toabout 1:100.Combinations of the above-noted ranges are possible (e.g., aratio of greater than or equal to about 1:1 and less than or equal toabout 10:1). Other ranges are also possible. In certain embodiments, theratio is about 1:1. In certain embodiments, the ratio is about 2:1. Incertain embodiments, the ratio is about 10:1.

In some embodiments, a composition and/or formulation may include theabove-noted ranges for the ratio of the weight of the drug to the weightof the one or more surface-altering agents during a formation processand/or a dilution process described herein. In certain embodiments, acomposition and/or formulation may include the above-noted ranges forthe ratio of the weight of the drug to the weight of the one or moresurface-altering agents in a final product.

The pharmaceutical agent may be present in the composition and/orformulation in any suitable amount, e.g., at least about 0.01 wt %, atleast about 0.1 wt %, at least about 1 wt %, at least about 5 wt %, atleast about 10 wt %, at least about 20 wt % of the composition and/orformulation. In some cases, the pharmaceutical agent may be present inthe composition and/or formulation at less than or equal to about 30 wt%, less than or equal to about 20 wt %, less than or equal to about 10wt %, less than or equal to about 5 wt %, less than or equal to about 2wt %, or less than or equal to about 1 wt %. Combinations of theabove-referenced ranges are also possible (e.g., present in an amount ofat least about 0.1 wt % and less than or equal to about 10 wt %). Otherranges are also possible. In certain embodiments, the pharmaceuticalagent is about 0.1-2 wt % of the composition and/or formulation. Incertain embodiments, the pharmaceutical agent is about 2-20 wt % of thecomposition and/or formulation. In certain embodiments, thepharmaceutical agent is about 0.2 wt % of the composition and/orformulation. In certain embodiments, the pharmaceutical agent is about0.4 wt % of the composition and/or formulation. In certain embodiments,the pharmaceutical agent is about 1 wt % of the composition and/orformulation. In certain embodiments, the pharmaceutical agent is about 2wt % of the composition and/or formulation. In certain embodiments, thepharmaceutical agent is about 5 wt % of the composition and/orformulation. In certain embodiments, the pharmaceutical agent is about10 wt % of the composition and/or formulation.

In one set of embodiments, a composition and/or formulation includes oneor more chelating agents. A chelating agent used herein refers to achemical compound that has the ability to react with a metal ion to forma complex through one or more bonds. The one or more bonds are typicallyionic or coordination bonds. The chelating agent can be an inorganic oran organic compound. A metal ion capable of catalyzing certain chemicalreactions (e.g., oxidation reactions) may lose its catalytic activitywhen the metal ion is bound to a chelating agent to form a complex.Therefore, a chelating agent may show preservative properties when itbinds to a metal ion. Any suitable chelating agent that has preservativeproperties can be used, such as phosphonic acids, aminocarboxylic acids,hydroxycarboxylic acids, polyamines, aminoalcohols, and polymericchelating agents. Specific examples of chelating agents include, but arenot limited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriaceticacid (NTA), diethylenetriaminepentacetic acid (DTPA),N-hydroxyethylethylene diaminetriacetic acid (HEDTA), tetraborates,triethylamine diamine, and salts and derivatives thereof. In certainembodiments, the chelating agent is EDTA. In certain embodiments, thechelating agent is a salt of EDTA. In certain embodiments, the chelatingagent is disodium EDTA.

A chelating agent may be present at a suitable concentration in acomposition and/or formulation including the coated particles describedherein. In certain embodiments, the concentration of the chelating agentis greater than or equal to about 0.0003 wt %, greater than or equal toabout 0.001 wt %, greater than or equal to about 0.003 wt %, greaterthan or equal to about 0.01 wt %, greater than or equal to about 0.03 wt%, greater than or equal to about 0.05 wt %, greater than or equal toabout 0.1 wt %, greater than or equal to about 0.3 wt %, greater than orequal to about 1 wt %, or greater than or equal to about 3 wt %. Incertain embodiments, the concentration of the chelating agent is lessthan or equal to about 3 wt %, less than or equal to about 1 wt %, lessthan or equal to about 0.3 wt %, less than or equal to about 0.1 wt %,less than or equal to about 0.05 wt %, less than or equal to about 0.03wt %, less than or equal to about 0.01 wt %, less than or equal to about0.003 wt %, less than or equal to about 0.001 wt %, or less than orequal to about 0.0003 wt %. Combinations of the above-noted ranges arepossible (e.g., a concentration of greater than or equal to about 0.01wt % and less than or equal to about 0.3 wt %). Other ranges are alsopossible. In certain embodiments, the concentration of the chelatingagent is about 0.001-0.1 wt %. In certain embodiments, the concentrationof the chelating agent is about 0.005 wt %. In certain embodiments, theconcentration of the chelating agent is about 0.01 wt %. In certainembodiments, the concentration of the chelating agent is about 0.05 wt%. In certain embodiments, the concentration of the chelating agent isabout 0.1 wt %.

In some embodiments, a chelating agent may be present in a compositionand/or formulation in one or more of the above-noted ranges during aformation process and/or a dilution process described herein. In certainembodiments, a chelating agent may be present in a composition and/orformulation in one or more of the above-noted ranges in a final product.

In some embodiments, an antimicrobial agent may be included in acomposition and/or formulation including the coated particles describedherein. An antimicrobial agent used herein refers to a bioactive agenteffective in the inhibition of, prevention of, or protection againstmicroorganisms such as bacteria, microbes, fungi, viruses, spores,yeasts, molds, and others generally associated with infections. Examplesof antimicrobial agents include cephaloporins, clindamycin,chlorampheanicol, carbapenems, minocyclines, rifampin, penicillins,monobactams, quinolones, tetracycline, macrolides, sulfa antibiotics,trimethoprim, fusidic acid, aminoglycosides, amphotericin B, azoles,flucytosine, cilofungin, bactericidal nitrofuran compounds,nanoparticles of metallic silver or an alloy of silver containing about2.5 wt % copper, silver citrate, silver acetate, silver benzoate,bismuth pyrithione, zinc pyrithione, zinc percarbonates, zincperborates, bismuth salts, parabens (e.g., methyl-, ethyl-, propyl-,butyl-, and octyl-benzoic acid esters), citric acid, benzalkoniumchloride (BAC), rifamycin, and sodium percarbonate.

An antimicrobial agent may be present at a suitable concentration in acomposition and/or formulation including the coated particles describedherein. In certain embodiments, the concentration of the antimicrobialagent may be greater than or equal to about 0.0003 wt %, greater than orequal to about 0.001 wt %, greater than or equal to about 0.003 wt %,greater than or equal to about 0.01 wt %, greater than or equal to about0.03 wt %, greater than or equal to about 0.1 wt %, greater than orequal to about 0.3 wt %, greater than or equal to about 1 wt %, orgreater than or equal to about 3 wt %. In certain embodiments, theconcentration of the antimicrobial agent may be less than or equal toabout 3 wt %, less than or equal to about 1 wt %, less than or equal toabout 0.3 wt %, less than or equal to about 0.1 wt %, less than or equalto about 0.03 wt %, less than or equal to about 0.01 wt %, less than orequal to about 0.003 wt %, less than or equal to about 0.001 wt %, orless than or equal to about 0.0003 wt %. Combinations of the above-notedranges are possible (e.g., a concentration of greater than or equal toabout 0.001 wt % and less than or equal to about 0.1 wt %). Other rangesare also possible. In certain embodiments, the concentration of theantimicrobial agent is about 0.001-0.05 wt %. In certain embodiments,the concentration of the antimicrobial agent is about 0.002 wt %. Incertain embodiments, the concentration of the antimicrobial agent isabout 0.005 wt %. In certain embodiments, the concentration of theantimicrobial agent is about 0.01 wt %. In certain embodiments, theconcentration of the antimicrobial agent is about 0.02 wt %. In certainembodiments, the concentration of the antimicrobial agent is about 0.05wt %.

In some embodiments, an antimicrobial agent may be present in acomposition and/or formulation in one or more of the above-noted rangesduring a formation process and/or a dilution process described herein.In certain embodiments, an antimicrobial agent may be present in acomposition and/or formulation in one or more of the above-noted rangesin a final product.

In some embodiments, a tonicity agent may be included in a compositionand/or formulation including the coated particles described herein. Atonicity agent used herein refers to a compound or substance that can beused to adjust the composition of a formulation to the desiredosmolarity range. In certain embodiments, the desired osmolarity rangeis an isotonic range compatible with blood. In certain embodiments, thedesired osmolarity range is hypotonic. In certain embodiments, thedesired osmolarity range is hypertonic. Examples of tonicity agentsinclude glycerin, lactose, mannitol, dextrose, sodium chloride, sodiumsulfate, sorbitol, saline-sodium citrate (SSC), and the like. In certainembodiments, a combination of one or more tonicity agents may be used.In certain embodiments, the tonicity agent is glycerin. In certainembodiments, the tonicity agent is sodium chloride.

A tonicity agent (such as one described herein) may be present at asuitable concentration in a composition and/or formulation including thecoated particles described herein. In certain embodiments, theconcentration of the tonicity agent is greater than or equal to about0.003 wt %, greater than or equal to about 0.01 wt %, greater than orequal to about 0.03 wt %, greater than or equal to about 0.1 wt %,greater than or equal to about 0.3 wt %, greater than or equal to about1 wt %, greater than or equal to about 3 wt %, greater than or equal toabout 10 wt %, greater than or equal to about 20 wt %, or greater thanor equal to about 30 wt %. In certain embodiments, the concentration ofthe tonicity agent is less than or equal to about 30 wt %, less than orequal to about 10 wt %, less than or equal to about 3 wt %, less than orequal to about 1 wt %, less than or equal to about 0.3 wt %, less thanor equal to about 0.1 wt %, less than or equal to about 0.03 wt %, lessthan or equal to about 0.01 wt %, or less than or equal to about 0.003wt %. Combinations of the above-noted ranges are possible (e.g., aconcentration of greater than or equal to about 0.1 wt % and less thanor equal to about 10 wt %). Other ranges are also possible. In certainembodiments, the concentration of the tonicity agent is about 0.1-1%. Incertain embodiments, the concentration of the tonicity agent is about0.5-3%. In certain embodiments, the concentration of the tonicity agentis about 0.25 wt %. In certain embodiments, the concentration of thetonicity agent is about 0.45 wt %. In certain embodiments, theconcentration of the tonicity agent is about 0.9 wt %. In certainembodiments, the concentration of the tonicity agent is about 1.2 wt %.In certain embodiments, the concentration of the tonicity agent is about2.4 wt %. In certain embodiments, the concentration of the tonicityagent is about 5 wt %. In certain embodiments, the tonicity agentcomprises glycerin in a concentration of about 0.1 wt %, about 0.2 wt %,about 0.3 wt %, about 0.4 wt %, about 0.5 wt %, about 0.6 wt %, about0.7 wt %, about 0.8 wt %, about 0.9 wt %, or about 1.0 wt %.

In some embodiments, a tonicity agent may be present in a compositionand/or formulation in one or more of the above-noted ranges during aformation process and/or a dilution process described herein. In certainembodiments, a tonicity agent may be present in a composition and/orformulation in one or more of the above-noted ranges in a final product.

In some embodiments, a composition and/or formulation described hereinmay have an osmolarity of at least about 0 mOsm/L, at least about 5mOsm/L, at least about 25 mOsm/L, at least about 50 mOsm/L, at leastabout 75 mOsm/L, at least about 100 mOsm/L, at least about 150 mOsm/L,at least about 200 mOsm/L, at least about 250 mOsm/L, at least about 310mOsm/L, or at least about 450 mOsm/L. In certain embodiments, acomposition and/or formulation described herein may have an osmolarityof less than or equal to about 450 mOsm/L, less than or equal to about310 mOsm/L, less than or equal to about 250 mOsm/L, less than or equalto about 200 mOsm/L, less than or equal to about 150 mOsm/L, less thanor equal to about 100 mOsm/L, less than or equal to about 75 mOsm/L,less than or equal to about 50 mOsm/L, less than or equal to about 25mOsm/L, or less than or equal to about 5 mOsm/L. Combinations of theabove-referenced ranges are also possible (e.g., an osmolarity of atleast about 0 mOsm/L and less than or equal to about 50 mOsm/L). Otherranges are also possible.

It is appreciated in the art that the ionic strength of a formulationcomprising particles may affect the polydispersity of the particles.Polydispersity is a measure of the heterogeneity of sizes of particlesin a formulation. Heterogeneity of particle sizes may be due todifferences in individual particle sizes and/or to the presence ofaggregation in the formulation. A formulation comprising particles isconsidered substantially homogeneous or “monodisperse” if the particleshave essentially the same size, shape, and/or mass. A formulationcomprising particles of various sizes, shapes, and/or masses is deemedheterogeneous or “polydisperse”.

The ionic strength of a formulation comprising particles may also affectthe colloidal stability of the particles. For example, a relatively highionic strength of a formulation may cause the particles of theformulation to coagulate and therefore may destabilize the formulation.In some embodiments, a formulation comprising particles is stabilized byrepulsive inter-particle forces. For example, the particles may beelectrically or electrostatically charged. Two charged particles mayrepel each other, preventing collision and aggregation. When therepulsive inter-particle forces weaken or become attractive, particlesmay start to aggregate. For instance, when the ionic strength of theformulation is increased to a certain level, the charges (e.g., negativecharges) of the particles may be neutralized by the oppositely chargedions present in the formulation (e.g., Na⁺ ions in solution). As aresult, the particles may collide and bond to each other to formaggregates (e.g., clusters or flocs) of larger sizes. The formedaggregates of particles may also differ in size, and thus thepolydispersity of the formulation may also increase. For example, aformulation comprising similarly-sized particles may become aformulation comprising particles having various sizes (e.g., due toaggregation) when the ionic strength of the formulation is increasedbeyond a certain level. In the course of aggregation, the aggregates maygrow in size and eventually settle to the bottom of the container, andthe formulation is considered colloidally unstable. Once the particlesin a formulation form aggregates, it is usually difficult to disrupt theaggregates into individual particles.

Certain formulations described herein show unexpected properties inthat, among other things, the presence of one or more ionic tonicityagents (e.g., a salt such as NaCl) in the formulations at certainconcentrations actually decreases or maintains the degree of aggregationof the particles present in the formulations, and/or does notsignificantly increase aggregation. See, for instance, Example 14. Incertain embodiments, the polydispersity of a formulation decreases, isrelatively constant, or does not change by an appreciable amount uponaddition of one or more ionic tonicity agents into the formulation.

For example, in some embodiments, the polydispersity of a compositionand/or formulation is relatively constant in the presence of added ionicstrength and/or when the added ionic strength of the composition and/orformulation is kept relatively constant or increased (e.g., during aformation and/or dilution process). In certain embodiments, when theionic strength increases by at least 50%, the polydispersity increasesby less than or equal to about 200%, less than or equal to about 150%,less than or equal to about 100%, less than or equal to about 75%, lessthan or equal to about 50%, less than or equal to about 30%, less thanor equal to about 20%, less than or equal to about 10%, less than orequal to about 3%, or less than or equal to about 1%. In certainembodiments, when the ionic strength is increased by at least 50%, thepolydispersity increases by greater than or equal to about 1%, greaterthan or equal to about 3%, greater than or equal to about 10%, greaterthan or equal to about 30%, or greater than or equal to about 100%.Combinations of the above-noted ranges are possible (e.g., an increasein polydispersity of less than or equal to 50% and greater than or equalto 1%). Other ranges are also possible.

The ionic strength of a formulation described herein may be controlled(e.g., increased) through a variety of means, such as the addition ofone or more ionic tonicity agents (e.g., a salt such as NaCl) to theformulation. In certain embodiments, the ionic strength of a formulationdescribed herein is greater than or equal to about 0.0005 M, greaterthan or equal to about 0.001 M, greater than or equal to about 0.003 M,greater than or equal to about 0.01 M, greater than or equal to about0.03 M, greater than or equal to about 0.1 M, greater than or equal toabout 0.3 M, greater than or equal to about 1 M, greater than or equalto about 3 M, or greater than or equal to about 10 M. In certainembodiments, the ionic strength of a formulation described herein isless than or equal to about 10 M, less than or equal to about 3 M, lessthan or equal to about 1 M, less than or equal to about 0.3 M, less thanor equal to about 0.1 M, less than or equal to about 0.03 M, less thanor equal to about 0.01 M, less than or equal to about 0.003 M, less thanor equal to about 0.001 M, or less than or equal to about 0.0005 M.Combinations of the above-noted ranges are possible (e.g., an ionicstrength of greater than or equal to about 0.01 M and less than or equalto about 1 M). Other ranges are also possible. In certain embodiments,the ionic strength of a formulation described herein is about 0.1 M. Incertain embodiments, the ionic strength of a formulation describedherein is about 0.15 M. In certain embodiments, the ionic strength of aformulation described herein is about 0.3 M.

In certain embodiments, the polydispersity of a formulation does notchange upon addition of one or more ionic tonicity agents into theformulation. In certain embodiments, the polydispersity does notsignificantly increase upon addition of one or more ionic tonicityagents into the formulation. In certain embodiments, the polydispersityincreases to a level described herein upon addition of one or more ionictonicity agents into the formulation.

The polydispersity of a formulation described herein may be measured bythe polydispersity index (PDI). The PDI is used to describe the width ofthe particle size distribution and is often calculated from a cumulantsanalysis of the dynamic light scattering (DLS) measured intensityautocorrelation function. The calculations for these parameters aredefined in the standards ISO 13321:1996 E and ISO 22412:2008. The PDI isdimensionless and, when measured by DLS, scaled such that values smallerthan 0.05 indicate a highly monodisperse sample while values greaterthan 0.7 indicate a very broad size distribution. In certainembodiments, the PDI of a formulation and/or composition describedherein is less than or equal to about 1, less than or equal to about0.9, less than or equal to about 0.8, less than or equal to about 0.7,less than or equal to about 0.6, less than or equal to about 0.5, lessthan or equal to about 0.4, less than or equal to about 0.3, less thanor equal to about 0.2, less than or equal to about 0.15, less than orequal to about 0.1, less than or equal to about 0.05, less than or equalto about 0.01, or less than or equal to about 0.005. In certainembodiments, the PDI of a formulation and/or composition describedherein is greater than or equal to about 0.005, greater than or equal toabout 0.01, greater than or equal to about 0.05, greater than or equalto about 0.1, greater than or equal to about 0.15, greater than or equalto about 0.2, greater than or equal to about 0.3, greater than or equalto about 0.4, greater than or equal to about 0.5, greater than or equalto about 0.6, greater than or equal to about 0.7, greater than or equalto about 0.8, greater than or equal to about 0.9, or greater than orequal to about 1. Combinations of the above-noted ranges are possible(e.g., a PDI of greater than or equal to about 0.1 and less than orequal to about 0.5). Other ranges are also possible. In certainembodiments, the PDI of a formulation is about 0.1. In certainembodiments, the PDI of a formulation is about 0.15. In certainembodiments, the PDI of a formulation is about 0.2.

In certain embodiments, the compositions and/or formulations describedherein may be highly dispersible and do not tend to form aggregates.Even when the particles do form aggregates, the aggregates may be easilybroken up into individual particles without rigorously agitating thecompositions and/or formulations.

Generally, it is desired that a formulation is sterile before or uponadministration to a subject. A sterile formulation is essentially freeof pathogenic microorganisms, such as bacteria, microbes, fungi,viruses, spores, yeasts, molds, and others generally associated withinfections. In some embodiments, compositions and/or formulationsincluding the coated particles described herein may be subject to anaseptic process and/or other sterilization process. An aseptic processtypically involves sterilizing the components of a formulation, finalformulation, and/or container closure of a drug product through aprocess such as heat, gamma irradiation, ethylene oxide, or filtrationand then combining in a sterile environment. In some cases, an asepticprocess is preferred. In other embodiments, terminal sterilization ispreferred.

Examples of other sterilization methods include radiation sterilization(e.g., gamma, electron, or x-ray radiation), heat sterilization, sterilefiltration, and ethylene oxide sterilization. The terms “radiation” and“irradiation” are used herein interchangeably. Unlike othersterilization methods, radiation sterilization has the advantage of highpenetrating ability and instantaneous effects, without the need tocontrol temperature, pressure, vacuum, or humidity in some instances. Incertain embodiments, the radiation used to sterilize the coatedparticles described herein is gamma radiation. Gamma radiation may beapplied in an amount sufficient to kill most or substantially all of themicrobes in or on the coated particles. The temperature of the coatedparticles described herein and the rate of radiation may be relativelyconstant during the entire gamma radiation period. Gamma irradiation maybe performed at any suitable temperature (e.g., ambient temperature,about 40° C., between about 30 to about 50° C.). Unless otherwiseindicated, measurements of gamma irradiation described herein refer toones performed at about 40° C.

In embodiments in which a sterilization process is used, it may bedesired that the process does not: (1) significantly change the particlesize of the coated particles described herein; (2) significantly changethe integrity of the active ingredient (such as a drug) of the coatedparticles described herein; and (3) generate unacceptable concentrationsof impurities during or following the process. In certain embodiments,the impurities generated during or following the process are degradantsof the active ingredient of the coated particles described herein. Forexample, when the active ingredient is loteprednol etabonate (LE),degradants of LE may include11β,17α-dihydroxy-3-oxoandrosta-1,4-diene-17-carboxylic acid (PJ-90),17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-1,4-diene-17β-carboxylicacid (PJ-91),17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-4-ene-17-carboxylicacid chloromethyl ester (tetradeca), and/or17α-[(ethoxycarbonyl)oxy]-3,11-dioxoandrosta-1,4-diene-17-carboxylicacid chloromethyl ester (11-keto), as shown in FIG. 22.

In certain embodiments, a process used to sterilize a composition and/orformulation described herein results in the presence of one or moredegradants in the formulation at less than or equal to about 10 wt %(relative to the weight of the undegraded drug), less than or equal toabout 3 wt %, less than or equal to about 2 wt %, less than or equal toabout 1.5 wt %, less than or equal to about 1 wt %, less than or equalto about 0.9 wt %, less than or equal to about 0.8 wt %, less than orequal to about 0.7 wt %, less than or equal to about 0.6 wt %, less thanor equal to about 0.5 wt %, less than or equal to about 0.4 wt %, lessthan or equal to about 0.3 wt %, less than or equal to about 0.2 wt %,less than or equal to about 0.15 wt %, less than or equal to about 0.1wt %, less than or equal to about 0.03 wt %, less than or equal to about0.01 wt %, less than or equal to about 0.003 wt %, or less than or equalto about 0.001 wt %. In some embodiments, the process results in adegradant in the formulation at greater than or equal to about 0.001 wt%, greater than or equal to about 0.003 wt %, greater than or equal toabout 0.01 wt %, greater than or equal to about 0.03 wt %, greater thanor equal to about 0.1 wt %, greater than or equal to about 0.3 wt %,greater than or equal to about 1 wt %, greater than or equal to about 3wt %, or greater than or equal to about 10 wt %. Combinations of theabove-referenced ranges are also possible (e.g., less than or equal toabout 1 wt % and greater than or equal to about 0.01 wt %). Other rangesare also possible.

In some embodiments, a composition and/or formulation subjected to gammairradiation includes a degradant having a concentration at one or moreof the above-noted ranges. In one set of embodiments, the drug isloteprednol etabonate and the degradant is PJ-90, PJ-91, tetradeca,and/or 11-keto. In certain embodiments, one or more, or each, of thedegradants is present in a composition and/or formulation at one or moreof the above-noted ranges (e.g., less than or equal to about 1 wt %,less than or equal to about 0.9 wt %, less than or equal to about 0.8 wt%, less than or equal to about 0.7 wt %, less than or equal to about 0.6wt %, less than or equal to about 0.5 wt %, less than or equal to about0.4 wt %, less than or equal to about 0.3 wt %, less than or equal toabout 0.2 wt %, or less than or equal to about 0.1 wt %). Other rangesare also possible.

In some embodiments, one or more additives are included in thecomposition and/or formulation to help achieve a relatively low amountof one or more degradants. For example, as described in Example 13, thepresence of glycerin in a loteprednol etabonate formulation resulted inrelatively low amounts of the degradant tetradeca after the formulationwas sterilized with gamma irradiation, compared to a loteprednoletabonate formulation that did not include glycerin.

When gamma irradiation is used in a sterilization process, thecumulative amount of the gamma radiation used may vary. In certainembodiments, the cumulative amount of the gamma radiation is greaterthan or equal to about 0.1 kGy, greater than or equal to about 0.3 kGy,greater than or equal to about 1 kGy, greater than or equal to about 3kGy, greater than or equal to about 10 kGy, greater than or equal toabout 30 kGy, greater than or equal to about 100 kGy, or greater than orequal to about 300 kGy. In certain embodiments, the cumulative amount ofthe gamma radiation is less than or equal to about 0.1 kGy, less than orequal to about 0.3 kGy, less than or equal to about 1 kGy, less than orequal to about 3 kGy, less than or equal to about 10 kGy, less than orequal to about 30 kGy, less than or equal to about 100 kGy, or less thanor equal to about 300 kGy. Combinations of the above-noted ranges arepossible (e.g., greater than or equal to about 1 kGy and less than orequal to about 30 kGy). Other ranges are also possible. In certainembodiments, multiple doses of radiation are utilized to achieve adesired cumulative radiation dosage.

The compositions and/or formulations described herein may have anysuitable pH values. The term “pH,” unless otherwise provided, refers topH measured at ambient temperature (e.g., about 20° C., about 23° C., orabout 25° C.). The compositions and/or formulations have, for example,an acidic pH, a neutral pH, or a basic pH and may depend on, forexample, where the compositions and/or formulations are to be deliveredin the body. In certain embodiments, the compositions and/orformulations have a physiological pH. In certain embodiments, the pHvalue of the compositions and/or formulations is at least about 1, atleast about 2, at least about 3, at least about 4, at least about 5, atleast about 6, at least about 6.2, at least about 6.4, at least about6.6, at least about 6.8, at least about 7, at least about 7.2, at leastabout 7.4, at least about 7.6, at least about 7.8, at least about 8, atleast about 8.2, at least about 8.4, at least about 8.6, at least about8.8, at least about 9, at least about 10, at least about 11, or at leastabout 12. In certain embodiments, the pH value of the compositionsand/or formulations is less than or equal to about 12, less than orequal to about 11, less than or equal to about 10, less than or equal toabout 9, less than or equal to about 8.8, less than or equal to about8.6, less than or equal to about 8.4, less than or equal to about 8.2,less than or equal to about 8, less than or equal to about 7.8, lessthan or equal to about 7.6, less than or equal to about 7.4, less thanor equal to about 7.2, less than or equal to about 7, less than or equalto about 6.8, less than or equal to about 6.6, less than or equal toabout 6.4, less than or equal to about 6.2, less than or equal to about6, less than or equal to about 5, less than or equal to about 4, lessthan or equal to about 3, less than or equal to about 2, or less than orequal to about 1. Combinations of the above-noted ranges are possible(e.g., a pH value of at least about 5 and less than or equal to about8.2). Other ranges are also possible. In certain embodiments, the pHvalue of the compositions and/or formulations described herein is atleast about 5 and less than or equal to about 8.

Methods, Compositions and Formulations for the Treatment of OcularConditions

The mammalian eye is a complex organ comprising an outer coveringincluding the sclera (the tough white portion of the exterior of theeye) and the cornea (the clear outer portion covering the pupil andiris). An exemplary schematic diagram of an eye is shown in FIG. 15A. Asshown illustratively in FIG. 15A in a medial cross section, fromanterior to posterior, an eye 100 comprises features including, withoutlimitation: a cornea 105, an iris 110 (a curtain-like feature that canopen and close in response to ambient light), a conjunctiva 115(composed of rare stratified columnar epithelium, covering the sclera,and lining the inside of the eyelids), a tear film 120 (which mayinclude an oil layer, a water layer, and a mucous layer (where themucous layer(s) has several functions such as serving as an anchor forthe tear film and helping it adhere to the eye), a corneal epithelium125 (several layers of cells covering the front of the cornea that actas a barrier to protect the cornea, resist the free flow of fluids fromthe tears, and prevent bacteria from entering), an anterior chamber 130(a hollow feature filled with a watery, clear fluid called the aqueoushumor 135 and bounded by the cornea in the front and the iris), a lens140 (a transparent, biconvex structure that, along with the cornea,which helps to refract light to be focused on the retina), a ciliarybody 145 (a circumferential tissue composed of ciliary muscles andciliary processes), a ciliary zonule 146 (a ring of fibrous strandsconnecting the ciliary body with the lens), a posterior chamber 148 (anarrow space bounded by the iris in front and by the ciliary zonule andthe ciliary body in back and also contains the aqueous humor, a retina150, a macula 155, a sclera 160, an optic nerve 165 (also known ascranial nerve, transmitting visual information from the retina to thebrain), a choroid 170, and avitreous chamber 175 (filled with a viscousfluid called the vitreous humor 180). The vitreous chamber comprisesapproximately ⅔ of the inner volume of the eye, while the anteriorchamber and posterior chamber comprise about ⅓ of the eye's innervolume.

As shown illustratively in FIG. 15B, there area number of mucus layersin the eye, present at a bulbar conjunctiva 116 (covering the eyeball,over the sclera, tightly bound to the underlying sclera, and moving withthe eyeball movements), a palpebral conjunctiva 117 (lining theeyelids), a fornix conjunctiva 118 (a loose and flexible tissue formingthe junction between the bulbar and palpebral conjunctivas and allowingthe free movement of the lids and eyeball), and the cornea. These mucuslayers form a complete surface that a topically administered medicationcontacts. Therefore, the topically administered medication typically hasto penetrate through these mucus layers in order to reach the variousunderneath eye tissues.

As shown illustratively in FIG. 15A, front of the eye, or anterior oranterior segment of the eye, depicted by a bracket 190, generallyincludes tissues or fluids that are located anterior to a posterior wall142 of a lens capsule 144 (a clear, membrane-like structure that iselastic and keeps the lens under constant tension) or ciliary muscles.The front of the eye includes, for example, the conjunctiva, the cornea,the iris, the tear film, the anterior chamber, the posterior chamber,the lens and the lens capsule, as well as blood vessels, lymphatics andnerves which vascularize, maintain or innervate an anterior ocularregion or site.

As shown illustratively FIG. 15A, the back of the eye, or posterior orposterior segment of the eye, depicted by a bracket 195, generallyincludes tissues or fluids that are located posterior to posterior wallof the lens capsule or ciliary muscles. The back of the eye includes,for example, the choroid, the sclera (in a position posterior to a planethrough the posterior wall of the lens capsule), the vitreous humor, thevitreous chamber, the retina, the macula, the optic nerve, and the bloodvessels and nerves which vascularize or innervate a posterior ocularregion or site.

As described in more detail below, in some embodiments, the particles,compositions and/or formulations described herein may be used todiagnose, prevent, treat or manage diseases or conditions at the back ofthe eye, such as at the retina, macula, choroid, sclera and/or uvea.

The retina is a 10-layered, delicate nervous tissue membrane of the eye,continuous with the optic nerve, that receives images of externalobjects and transmits visual impulses through the optic nerve to thebrain. The retina is soft and semitransparent and contains rhodopsin. Itconsists of the outer pigmented layer and the nine-layered retinaproper. These nine layers, starting with the most internal, are theinternal limiting membrane, the stratum opticum, the ganglion celllayer, the inner plexiform layer, the inner nuclear layer, the outerplexiform layer, the outer nuclear layer, the external limitingmembrane, and the layer of rods and cones. The outer surface of theretina is in contact with the choroid; the inner surface with thevitreous body. The retina is thinner anteriorly, where it extends nearlyas far as the ciliary body, and thicker posteriorly, except for a thinspot in the exact center of the posterior surface where focus is best.The photoreceptors end anteriorly in the jagged ora serrata at theciliary body, but the membrane of the retina extends over the back ofthe ciliary processes and the iris. The retina becomes clouded andopaque if exposed to direct sunlight.

The macula or macula lutea is an oval-shaped highly pigmented yellowspot near the center of the retina of the human eye. It has a diameterof around 5 mm and is often histologically defined as having two or morelayers of ganglion cells. Near its center is the fovea, a small pit thatcontains the largest concentration of cone cells in the eye and isresponsible for central, high resolution vision. The macula alsocontains the parafovea and perifovea. Because the macula is yellow incolor it absorbs excess blue and ultraviolet light that enter the eye,and acts as a natural sunblock (analogous to sunglasses) for this areaof the retina. The yellow color comes from its content of lutein andzeaxanthin, which are yellow xanthophyll carotenoids, derived from thediet. Zeaxanthin predominates at the macula, while lutein predominateselsewhere in the retina. There is some evidence that these carotenoidsprotect the pigmented region from some types of macular degeneration.Structures in the macula are specialized for high acuity vision. Withinthe macula are the fovea and foveola which contain a high density ofcones (photoreceptors with high acuity).

The choroid, also known as the choroidea or choroid coat, is thevascular layer of the eye, containing connective tissue, and lyingbetween the retina and the sclera. The human choroid is thickest at thefar extreme rear of the eye (at 0.2 mm), while in the outlying areas itnarrows to 0.1 mm. The choroid provides oxygen and nourishment to theouter layers of the retina. Along with the ciliary body and iris, thechoroid forms the uveal tract.

The sclera refers to the tough inelastic opaque membrane covering theposterior five sixths of the eyebulb. It maintains the size and form ofthe bulb and attaches to muscles that move the bulb. Posteriorly it ispierced by the optic nerve and, with the transparent cornea, makes upthe outermost of three tunics covering the eyebulb.

The uvea refers to the fibrous tunic beneath the sclera that includesthe iris, the ciliary body, and the choroid of the eye.

Ophthalmic therapy may be performed by topically administeringcompositions, such as eye drops, to the exterior surface of the eye. Eyedrops administration is by far the most desired route of drug deliveryto the eye due to its convenience, non-invasive nature, localizedaction, and relative patient comfort. However, drugs administered to theeye topically as solutions (e.g., ophthalmic solutions) may be rapidlycleared from the eye's surface by drainage and lachrymation. Drugsadministered to the eye topically as particles (e.g., ophthalmicsuspensions) are typically trapped by the mucus layer or tear film inthe eye. The eye's natural clearance mechanisms remove the speciestrapped in such a layer, and hence, drugs trapped in this layer are alsorapidly cleared. As a result, achieving desired drug levels in the eye,especially in the posterior portions of the eye, such as the posteriorsclera, the uveal tract (located in the vascular middle layer of theeye, constituting the iris, ciliary body, and choroids), the vitreous,the choroid, the retina, and the optic nerve head (ON H), or even theinner portions of the cornea, by topical route of administration isoften difficult.

For example, cornea and conjunctiva are naturally covered by a 3-40 μmlayer of mucus. As shown illustratively in FIG. 15C, the outer layer iscomprised of secreted mucins 310 (cleared rapidly by mucin turnover andblinking), whose primary role is to trap and eliminate allergens,pathogens, and debris (including drug particles) from the aqueous layer305 of the eye. The inner layer (thickness up to 500 nm) is formed bymucins tethered to epithelium 315 (glycocalyx), which protects theunderlying tissue from abrasive stress and are cleared less rapidly.Without wishing to be bound by theory, it is believed that conventionalparticles (CP; i.e., non-MPP) are trapped in the outer mucus layer andare readily cleared from the ocular surface. Thus, the conventionalparticles may be cleared before the drugs contained in the particles canbe transported to other portions of the eye (e.g., by diffusion or othermechanisms). In contrast, the particles described herein (e.g., MPP) mayavoid adhesion to secreted mucins, and thus may penetrate the peripheralmucus layer and reach the slow-clearing glycocalyx, thereby prolongingparticle retention and sustaining drug release (FIG. 15C). This suggeststhat the particles described herein may deliver drugs to underlyingtissues (cornea, conjunctiva, etc.) much more efficiently than CPtrapped in outer mucus. Furthermore, the formulations described hereinmay create an even coverage of particles and/or pharmaceutical agentover the whole surface of the eye, where a conventional formulationwithout the coatings described herein may not spread as evenly due totheir immobilization in mucus. Therefore, the formulations describedherein may enhance efficacy by more uniform coverage. This in turn,along with higher concentrations, may enhance penetration through mucus.

Moreover, the use of the particles described herein for topicaladministration may address some of the challenges associated with othermodes of delivery to the eye, such as injection methods and the use oftopical gels or inserts. Injection methods may be effective fordelivering drugs to the posterior portions of the eye, but such methodsare invasive and may not be desirable. Other methods of delivery, suchas topical gels and/or various inserts, that may help to deliver drug tothe eye are less desirable from the patient comfort perspective.

There are other challenges associated with topical administration to theeye. The absorption of pharmaceutical agents into the eye is severelylimited by some protective mechanisms that ensure the proper functioningof the eye, and by other concomitant factors, for example: drainage ofthe instilled solutions; lacrimation and tear turnover; metabolism; tearevaporation; non-productive absorption/adsorption; limited corneal areaand poor corneal permeability; and binding by the lacrimal proteins.Therefore, eye drops that can achieve and maintain a high concentrationof the pharmaceutical agent for an extended duration at the eye'ssurface would be desirable.

For example, the drainage of the administered dose via the nasolacrimalsystem into the nasopharynx and the gastrointestinal tract takes placewhen the volume of fluid in the eye exceeds the normal lacrimal volumeof seven to 10 microlitres. Thus, the portion of the instilled dose (oneto two drops, corresponding to 50-100 microlitres) that is noteliminated by spillage from the palpebral fissure may be drained quicklyand the contact time of the dose with the absorbing surfaces (cornea andsclera) may be reduced to, for example, a maximum of two minutes.

The lacrimation and the physiological tear turnover (e.g., 16% perminute in humans in normal conditions) can be stimulated and increasedby the instillation even of mildly irritating solutions. The net resultis a dilution of the applied medication and an acceleration of the lossof the pharmaceutical agent.

Topically administered drug-loaded micro- and nanoparticles have thepotential to prolong ocular retention and increase local bioavailabilityof drugs without causing the discomfort associated with other sustainedrelease formulations such as gels, ointments and inserts. However, amajor barrier for such particles is the mucus layer at ocular surfaces(e.g., at the palpebral conjunctiva, bulbar conjunctiva, and cornea;FIG. 15B). This mucus, whose natural role is to clear debris andallergens, effectively traps and rapidly removes virtually all foreignparticles, including drug-loaded nanoparticles, from the ocular surface.To prolong drug retention at the ocular surface and deliver the drugcloser to the underlying tissue, drug carriers/particles may need toavoid adhesion to the rapidly-clearing mucus. Therefore, drugcarriers/particles that can avoid or have reduced adhesion to mucuswould be desirable.

For an effective amount of a pharmaceutical agent to be delivered intoan eye, high doses and/or frequent dosages may be used. However, highdoses of a pharmaceutical agent increase the risk of local and systemicside effects. Moreover, frequent administration is not desirable becauseof its inconvenience caused to the patient, often resulting in poorcompliance. Therefore, improving the mucus penetration of thepharmaceutical agent by using appropriate formulations, such as thosedescribed herein, becomes advantageous because an effectiveconcentration of the pharmaceutical agent in the eye can be achievedwithout the need to use high and/or frequent doses.

Moreover, without wishing to be bound by theory, it is believed thattopically administered pharmaceutical agents may be transported to theback of the eye through one or more of three main pathways: 1) thetrans-vitreous trans corneal diffusion followed by entry into vitreousand subsequent distribution to ocular tissues (FIG. 16, pathway 205); 2)the uvea-scleral route, i.e., trans-corneal diffusion, passage throughthe anterior chamber, and drainage via the aqueous humor to theuvea-scleral tissue towards the posterior tissues (FIG. 16, pathway210); and 3) the periocular route, i.e., permeation through theconjunctiva to access the periocular fluid of the tenon, diffusionaround the sclera followed by diffusion across the sclera, choroid, andretina (FIG. 16, pathway 215) (Uday B. Kompella and Henry F. Edelhauser;Drug Product Development for the Back of the Eye, 1^(st) Ed.; Springer;2011). Due to anatomic membrane barriers (i.e. cornea, conjunctiva andsclera) and the lachrymal drainage, it can be quite challenging toobtain therapeutic drug concentrations in the posterior parts of the eyeafter topical drug administration. Reaching the posterior part of theeye is even more challenging task because of the anatomical andphysiological barriers associated with this part of the eye. Since thosebarriers cannot be altered with non-invasive methods, improvements inophthalmic compositions and formulations that would increase the ocularbioavailability and address other challenges of topical administrationto the eye would be beneficial.

The urgency to develop such formulations can be inferred from the factthat the leading causes of vision impairment and blindness areconditions linked to the posterior segment of the eye. These conditionsmay include, without limitation, age-related ocular degenerativediseases such as, age-related macular degeneration (AMD), proliferativevitreoretinopathy (PVR), retinal ocular condition, retinal damage,macular edema (e.g., cystoid macular edema (CME) or (diabetic macularedema (DME)), and endophthalmitis. Glaucoma, which is often thought ofas a condition of the anterior chamber affecting the flow (and thus, theintraocular pressure (IOP)) of aqueous humor, also has a posteriorsegment component. In fact, certain forms of glaucoma are notcharacterized by high IOP, but mainly by retinal degeneration alone.

In certain embodiments, these and other conditions may be treated,diagnosed, prevented, or managed using the mucus-penetrating particles,compositions and formulations described herein. For example, topicaladministration of eye drops containing mucus-penetrating particles maybe used to effectively deliver an anti-AMD drug to the back of the eyeand treat AMD without subjecting the patient to an invasive proceduresuch as an intravetreal injection. Or, for example, topicaladministration of eye drops containing mucus-penetrating particlesloaded with an anti-inflammatory drug (e.g. corticosteroid or NSIAD) canbe used for the treatment of ocular inflammation at a reduced dosingfrequency.

The particles, compositions, and methods described herein may addressthe challenges described herein associated with the delivery ofpharmaceutical agents to the anterior and/or posterior portions of theeye at least in part due to the mucus-penetrating properties of theparticles. Without wishing to be bound by theory, it is believed thatthe particles described herein having mucus-penetrating properties areable to avoid adhesion to, and effectively penetrate through, the mucuscoating the eye. As the particles penetrate through the mucus layer ofan eye tissue (e.g., palpebral conjunctiva, bulbar conjunctiva, cornea,or tear film), they avoid rapid clearance by the body's naturalclearance mechanisms and achieve prolonged retention at the front of theeye. The particles may then be dissolved and/or may release apharmaceutical agent as the particles and/or pharmaceutical agents aretransported towards the back of the eye, e.g., by one of the mechanismsdescribed in FIG. 16. By contrast, particles or drugs that are not mucuspenetrating may adhere to mucus, and may be rapidly cleared by thebody's natural clearance mechanisms such that insufficient amounts ofthe particles or drugs remain at the front of the eye shortly afteradministration. Thus, relatively low amounts of the particles or drugsmay be available for being transported to the back of the eye (e.g., bydiffusion or other mechanisms). For example, as described in more detailin Examples 3 and 8, the marketed ophthalmic suspension Lotemax®, whichincludes particles of the pharmaceutical agent loteprednol etabonate(LE) that do not effectively penetrate mucus, was compared with LEparticles having a coating of Pluronic® F127. This drug is typicallyused for treating inflammation of tissues at the front of the eye.Surprisingly, when particles of the pharmaceutical agent LE were coatedwith a coating of Pluronic® F127, Tween 80®, or certain PVAs renderingthem mucus-penetrating, not only was delivery to the ocular surface atthe front of the eye (e.g., cornea, iris/ciliary body, aqueous humor)enhanced markedly as described in Examples 3 and 34, but delivery to themiddle and back of the eye (e.g., retina, choroid, and sclera) was alsoenhanced as described in Example 8. These results were unexpected, inparticular, because LE has not been previously shown to penetrate to theback of the eye when dosed topically as an eye drop. Moreover,conventional wisdom would suggest that LE particles coated withPluronic® F127 would be rapidly washed away from the ocular surface bylachrymation since many have previously reported that nanosized drugparticles are highly soluble in the solution in which they are containedand, thus, are expected to behave more like a conventional solutionincapable of sustained release. It should be appreciated that while muchof the description herein refers to treating, diagnosing, preventing, ormanaging tissues in the posterior portions of the eye or the back of theeye, the methods, compositions and formulations described herein are notso limited, and that other portions of the eye may benefit from themethods, compositions and formulations described herein.

In one aspect, the present invention provides a formulation orpharmaceutical composition for administration to an eye of a subject,comprising a plurality of coated particles, wherein the coated particlescomprise a particle core comprising loteprednol etabonate and a coatingcomprising poloxamer 407 (Pluronic F127) surrounding the particle core,and pharmaceutically acceptable excipients, wherein saidpharmaceutically acceptable excipients comprise glycerin, sodiumcholoride, disodium edetate, citric acid, and benzalkonium chloride. Theformulation or pharmaceutical composition may further comprise one ormore buffering agents, wherein the bufferent agent is trisodium citrate,citric acid, or a combination thereof.

In one aspect, the present invention provides a formulation orpharmaceutical composition of loteprednol etabonate, wherein theformulation or pharmaceutical composition comprises 0.25% w/vloteprednol etabonate, 0.125% w/v poloxamer 407, and 0.6% w/v glycerin.The formulation may also comprise sodium chloride, trisodium citratedihydrate, disodium edetate dihydrate, citric acid, and benzalkoniumchloride. The formulation or pharmaceutical composition may furthercomprise purified water. In one embodiment, the formulation orpharmaceutical composition is a suspension.

In another aspect, the present invention provides a formulation orpharmaceutical composition of loteprednol etabonate, wherein theformulation or pharmaceutical composition comprises 1.0% w/v loteprednoletabonate, 0.5% w/v poloxamer 407, and 0.6% w/v glycerin. Theformulation may also comprise sodium chloride, trisodium citratedihydrate, disodium edetate dihydrate, citric acid, and benzalkoniumchloride. The formulation or pharmaceutical composition may furthercomprise purified water. In one embodiment, the formulation orpharmaceutical composition is a suspension.

Additionally, described in Examples 21 and 29-33 are particles andcompositions including an RTK inhibitor (e.g., sorafenib, linifanib,MGCD-265, pazopanib, cediranib, and axitinib), which demonstrateenhanced exposure of the RTK inhibitor at the back of the eye.

Portions of the eye that may be targeted or treated by the methods,compositions, and formulations described herein are now described inmore detail.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to target and/or treat theconjunctiva of a subject. The conjunctiva refers to the mucous membranelining the inner surfaces of the eyelids and anterior part of thesclera. The palpebral conjunctiva lines the inner surface of the eyelidsand is thick, opaque, and highly vascular. The bulbar conjunctiva isloosely connected, thin, and transparent, covering the sclera or theanterior third of the eye.

In certain embodiments, the methods, particles, compositions, and/orformulations described herein may be used to target and/or treat the allor portions of the cornea of a subject. The cornea refers to the convex,transparent anterior part of the eye, comprising one sixth of theoutermost tunic of the eye bulb. It allows light to pass through it tothe lens. The cornea is a fibrous structure with five layers: theanterior corneal epithelium, continuous with that of the conjunctiva;the anterior limiting layer (Bowman's membrane); the substantialpropria; the posterior limiting layer (Descemet's membrane); and theendothelium of the anterior chamber (keratoderma). It is dense, uniformin thickness, and nonvascular, and it projects like a dome beyond thesclera, which forms the other five sixths of the eye's outermost tunic.The degree of corneal curvature varies among different individuals andin the same person at different ages; the curvature is more pronouncedin youth than in advanced age.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to target and/or treatportions within the posterior portion or back of the eye, such as theretina, the choroid, and/or the sclera, of a subject. Starting from theinside of the eye and going towards the back, the three main layers atthe back of the eye are the retina, which contains the nerves; thechoroid, which contains the blood supply; and the sclera, which is thewhite of the eye.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage an ocular condition, i.e., a disease, ailment, or conditionthat affects or involves the eye or one or more of the parts or regionsof the eye. Broadly speaking, the eye includes the eyeball and thetissues and fluids which constitute the eyeball, the periocular muscles(such as the oblique and rectus muscles) and the portion of the opticnerve which is within or adjacent to the eyeball.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage an ocular condition at the front of the eye of a subject. Ingeneral, a front of the eye (or anterior or anterior segment) ocularcondition is a disease, ailment or condition which affects or involves atissue or a fluid at the front of the eye, as described herein. A frontof the eye ocular condition includes a disease, ailment or condition,such as for example, post-surgical inflammation; uveitis; infections;aphakia; pseudophakia; astigmatism; blepharospasm; blepharitis;cataract; conjunctival diseases; conjunctivitis; corneal diseases;corneal ulcer; dry eye syndromes; eyelid diseases; lacrimal apparatusdiseases; lacrimal duct obstruction; myopia; presbyopia; pupildisorders; corneal neovascularization; refractive disorders andstrabismus. Glaucoma can be considered to be a front of the eye ocularcondition in some embodiments because a clinical goal of glaucomatreatment can be to reduce a hypertension of aqueous fluid in theanterior chamber of the eye (i.e., reduce intraocular pressure).

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage an ocular condition at the back of the eye of a subject. Ingeneral, a back of the eye or posterior ocular condition is a disease,ailment, or condition which primarily affects or involves a tissue orfluid at the back of the eye, as described herein. A posterior ocularcondition can include a disease, ailment, or condition, such asintraocular melanoma; acute macular neuroretinopathy; Behcet's disease;choroidal neovascularization; uveitis; diabetic uveitis; histoplasmosis;infections, such as fungal or viral-caused infections; maculardegeneration, such as acute macular degeneration, non-exudative agerelated macular degeneration and exudative age related maculardegeneration; edema, such as macular edema (e.g., cystoid macular edema(CME) and diabetic macular edema (DME)); multifocal choroiditis; oculartrauma which affects a posterior ocular site or location; ocular tumors;retinal disorders, such as central retinal vein occlusion, diabeticretinopathy (including proliferative diabetic retinopathy),proliferative vitreoretinopathy (PVR), retinal arterial occlusivedisease, retinal detachment, uveitic retinal disease; sympatheticopthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uveal diffusion; aposterior ocular condition caused by or influenced by an ocular lasertreatment; posterior ocular conditions caused by or influenced by aphotodynamic therapy, photocoagulation, radiation retinopathy,epiretinal membrane disorders, branch retinal vein occlusion, anteriorischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction,retinitis pigmentosa, retinoblastoma, and glaucoma. Glaucoma can beconsidered a posterior ocular condition in some embodiments because thetherapeutic goal is to prevent the loss of or reduce the occurrence ofloss of vision due to damage to or loss of retinal cells or optic nervecells (i.e., neuroprotection).

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage dry eye in a subject. In one embodiment, the methods,particles, compositions, and/or formulations comprising loteprednoletabonate described herein may be used to treat, diagnose, prevent, ormanage dry eye in a subject. Dry eye is a condition in which there areinsufficient tears to lubricate and nourish the eye. Tears are necessaryfor maintaining the health of the front surface of the eye and forproviding clear vision. People with dry eyes either do not produceenough tears or have a poor quality of tears. Dry eye is a common andoften chronic problem, particularly in older adults.

With each blink of the eyelids, tears are spread across the frontsurface of the eye, known as the cornea. Tears provide lubrication,reduce the risk of eye infection, wash away foreign matter in the eye,and keep the surface of the eyes smooth and clear. Excess tears in theeyes flow into small drainage ducts, in the inner corners of theeyelids, which drain in the back of the nose. Tears are produced byseveral glands (e.g., lacrimal gland) in and around the eyelids. Tearproduction tends to diminish with age, with various medical conditions,or as a side effect of certain medicines. Environmental conditions suchas wind and dry climates can also affect tear volume by increasing tearevaporation. When the normal amount of tear production decreases ortears evaporate too quickly from the eyes, symptoms of dry eye candevelop.

The most common form of dry eyes is due to an inadequate amount of thewater layer of tears. This condition, called keratoconjunctivitis sicca(KCS), is also referred to as dry eye syndrome.

Treatments for dry eyes aim to restore or maintain the normal amount oftears in the eye to minimize dryness and related discomfort and tomaintain eye health. These goals may be achieved through differentpathways, such as increasing the lacrimal glands' tear production,regulating conjunctiva's mucin production, and suppressing inflammationof eye tissues. For example, Restasis® (0.05% cyclosporine) is animmunosuppressant that lowers the activity of T cells in the conjunctivaand lacrimal gland. Challenges in developing new treatment of dry eyesinclude identifying underlying diseases and causes, length of time tosee results (3-6 months), and the fact that treatment may only work in10-15% of dry eye population. Drug delivery may also be a challenge.Although the eye tissues targeted in the treatment of dry eyes are atthe front of the eye, a fraction of a topically administeredpharmaceutical agent may still be immobilized by the mucus in theconjunctiva, tear film, and cornea). In some embodiments, the particles,formulations, and compositions described herein may address these issuesby facilitating effective delivery of pharmaceutical agents to theappropriate tissues, promoting more even and/or wide-spread coverage ofthe particles across the eye surface, and/or avoiding or minimizingclearance of the particle/pharmaceutical agent.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage inflammation in the eye of a subject. Inflammation isassociated with a variety of ocular disorders. Inflammation may alsoresult from a number of ophthalmic surgical procedures, includingcataract surgery. Corticosteroids are often used as ocularanti-inflammatory agents, however, they typically require frequentdosing.

To prevent post-surgical inflammation, steroids or NSAIDs (non-steroidalanti-inflammatory drugs) may be given prophylactically. Currenttreatment of post-surgical inflammation includes steroids (e.g.,Lotemax® (0.5% loteprednol etabonate), Durezol® (0.05% difluprednate),Pred Mild® (0.12% prednisolone acetate), and Omnipred® (1% prednisoloneacetate)) and NSAIDs (e.g., Bromday® (0.09% bromfenac), Nevanac® (0.1%nepafenac), Acular LS® (0.4% ketorolac tromethamine), Acuvail® (0.45%ketorolac tromethamine)), Toradol® (ketorolac tromethamine), Sprix®(ketorolac tromethamine), Voltaren® (0.1% diclofenac), Aclonac®(diclofenac), and Cataflam® (diclofenac). One of the greatest challengesfor the treatment of post-surgical inflammation is compliance inasmuchas, due to the rapid clearance of eye drops from the surface of the eye,most currently marketed steroid or NSAID eye drops must be administeredmultiple times a day to achieve and sustain therapeutic effect. In someembodiments, the particles, compositions, and/or formulations describedherein may include one or more of these steroidal pharmaceutical agents.For example, as described in more detail in the Examples, particles ofLoteprednol Etabonate, the ingredient of Lotemax®, that included certainpolymeric coatings described herein produced markedly higher drug levelsin various ocular tissues in New Zealand white rabbits, compared to anequivalent dose of the commercial formulation that did not include asuitable polymeric coating. This data suggests that the coated particlesmay be administered fewer times a day to achieve and sustain therapeuticeffect compared to commercial formulations.

A number of topical NSAID formulations (e.g., Bromday® (0.09%bromfenac)) are available in the market. Table 17 provides a list ofthese formulations, their respective trade names, active pharmaceuticalingredients (APIs), dosing concentration, and dosing frequency. Themajority of these formulations (i.e., Bromday®, Flurbiprofen®, Acular®,and Voltaren®) are supplied as solutions in which the active ingredientis completely dissolved.

It has been found that bromfenac is susceptible to degradation insolution via lactam formation, especially below neutral pH (Table 18).Data in Table 18 show that more degradant of bromfenac was observed whenthe pH of an aqueous solution containing bromfenac sodium was lowered(e.g., from pH 7.8 to 5.8).

To enhance topical delivery of bromfenac, which in turn may translateinto a lower dose for improved safety or enhanced therapy for conditionsin the middle and back of the eye, it may be desirable to formulatebromfenac as a suspension of MPPs comprising a bromfenac core.Additionally, formulating bromfenac as a suspension of MPPs may allowfor an increase in the concentration of bromfenac in the formulationwithout substantially increasing the concentration of degradants (e.g.,compared to an aqueous solution of bromfenac). However, it is difficultto formulate bromfenac sodium as a solid or crystalline particle due toits relatively high water solubility. Bromfenac free acid (bromfenac FA)may also be difficult to develop into a shelf-stable MPP suspensionformulation in some embodiments, e.g., due to significant degradation ofbromfenac FA in the presence of aqueous Pluronic® F127 (Table 19).

TABLE 17 Currently available products featuring topically-deliveredNSAIDs. Treatment Trade Concentration frequency Dosage name ManufacturerAPI of API (% w/v) (drops per day) form Bromday ® Bausch & bromfenac0.09 1 solution Lomb sodium Ocufen ® Allergan flurbiprofen 0.03 4solution Acular ®, Allergan ketorolac 0.4-0.5 2-4 solution Acular LS ®,tromethamine Acuvail ® Voltaren ® Novartis diclofenac 0.1 4 solutionsodium Nevanac ® Alcon nepafenac 0.1 3 suspension The products listedare prescribed for administration post-surgery, with the exception ofOcufen ® which is administered within 2 hr prior to surgery.

TABLE 18 Chemical degradation of bromfenac sodium at various pH.* pH %peak area 5.8 3.16 6.8 0.05 7.8 0 *The chemical stability of bromfenacsodium was determined as % chromatographic peak area for the lactamdegradant of bromfenac after 0.02% aqueous solutions of bromfenac sodiumwere stored for 5 days at room temperature.

TABLE 19 Chemical degradation of bromfenac free acid in unbufferedaqueous suspensions in the presence of Pluronic ® F127.* Concentrationof Concentration Pluronic ® F127 of Bromfenac (% w/v) FA (% w/v) % peakarea 0.50 0.5 12.0 (day 14) 5 5 10.2 (day 5)  *The chemical stability ofbromfenac free acid was determined as % chromatographic peak area forthe lactam degradant of bromfenac after an aqueous suspension ofbromfenac free acid was stored for 5 or 14 days at room temperature.

As described herein, it is desirable to develop a composition comprisingan NSAID (e.g., bromfenac, diclofenac, ketorolac, or a salt thereof)that is stable at a suitable pH for topical administration to the eye.In some embodiments, such compositions include solid or crystallineparticles of bromfenac, diclofenac, ketorolac, or a salt thereof, thatcan effectively penetrate mucus. The particles may include one or moresurface-altering agents described herein (e.g., a poloxamer, apolysorbate (e.g., Tween 80®), PVA) that can reduce mucoadhesion of theparticles.

In some embodiments, the particles, compositions, and/or formulationsdescribed herein include a divalent metal salt of bromfenac, such as adivalent metal salt of bromfenac. For instance, the divalent metal saltof bromfenac may be relatively water insoluble and may include, forexample, bromfenac beryllium, bromfenac magnesium, bromfenac calcium,bromfenac strontium, bromfenac barium, bromfenac zinc, or bromfenaccopper(II). In some embodiments, the particles including a divalentmetal salt of bromfenac may have an aqueous solubility in a rangedescribed herein (e.g., at least about 0.001 mg/mL and less than orequal to about 1 mg/mL).

In certain embodiments, the particles, compositions, and/or formulationsdescribed herein include diclofenac FA. In certain embodiments, theparticles, compositions, and/or formulations described herein include ametal salt of diclofenac, such as an alkaline earth metal salt ofdiclofenac. In certain embodiments, the particles, compositions, and/orformulations described herein include ketorolac FA. In certainembodiments, the particles, compositions, and/or formulations describedherein include a metal salt of ketorolac, such as an alkaline earthmetal salt of ketorolac. Trivalent metal salts of such compounds arealso possible.

The divalent metal salts of bromfenac described herein (e.g., bromfenaccalcium) are less water soluble and more hydrophobic than bromfenacsodium and/or other monovalent salts of bromfenac. For example, theaqueous solubility of bromfenac calcium at 25° C. is about 0.15 mg/mL.Compared to the more water soluble and hydrophilic bromfenac sodium, thedivalent metal salts of bromfenac may be more suitable to be processedinto MPPs using the methods described herein (e.g., milling and/orprecipitation). The divalent metal salts of bromfenac are present in theMPPs mostly in solid (e.g., crystalline) form and, therefore, may beless prone to degradation and more chemically stable. Additionally,relatively high concentrations of the divalent metal salts of bromfenacin a composition and/or formulation including MPPs of the divalent metalsalts of bromfenac are not limited by the aqueous solubility and/or theformation of degradants of the divalent metal salts of bromfenac.Therefore, the particles, compositions, and/or formulations describedherein comprising a divalent metal salt of bromfenac may allow forhigher concentrations of bromfenac in the compositions or formulationscompared to the free acid form which is dissolved in solution. In someembodiments, such particles, compositions, and/or formulations allow forhigher concentrations of bromfenac in ocular tissues afteradministration to the eye.

For similar reasons discussed herein with respect to bromfenac calciumand the free acid form of bromfenac, the less water soluble andhydrophilic diclofenac FA and a metal salt thereof (e.g., divalent ortrivalent metal salts) may be more suitable for being processed intoparticles, compositions, and/or formulations that are mucus penetratingcompared to diclofenac sodium and/or other monovalent salts ofdiclofenac. Similarly, ketorolac FA and a metal salt thereof (e.g.,divalent or trivalent metal salts), which are less water soluble andhydrophilic than ketorolac tromethamine and/or other monovalent salts ofketorolac, may be formed into mucus penetrating particles, compositions,and/or formulations. Moreover, since diclofenac FA or ketorolac FA, or adivalent or trivalent metal salt thereof, may not be limited by theiraqueous solubilities, these compounds may be present in a higherconcentration in the particles, compositions, and/or formulationsdescribed herein compared to aqueous formulations of diclofenac sodiumor ketorolac tromethamine, respectively.

In certain embodiments, a pharmaceutical agent described herein (e.g.,an NSAID such as a divalent metal salt of bromfenac (e.g., bromfenaccalcium), diclofenac FA, a metal salt of diclofenac (e.g., divalent ortrivalent metal salts), ketorolac FA, or a metal salt of ketorolac(e.g., divalent or trivalent metal salts); a receptor tyrosine kinase(RTK) inhibitor, such as sorafenib, linifanib, MGCD-265, pazopanib,cediranib, and axitinib; or a corticosteroid, such as LE) is present ina composition and/or formulation described herein at at least about0.001%, at least about 0.003%, at least about 0.01%, at least about0.02%, at least about 0.05%, at least about 0.1%, at least about 0.2%,at least about 0.3%, at least about 0.4%, at least about 0.5%, at leastabout 0.6%, at least about 0.8%, at least about 1%, at least about 1.5%,at least about 2%, at least about 3%, at least about 4%, at least about5%, at least about 6%, at least about 8%, at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, or at least about50%, w/v. In certain embodiments, the pharmaceutical agent is present ina composition and/or formulation described herein at less than or equalto about 50%, less than or equal to about 40%, less than or equal toabout 30%, less than or equal to about 20%, less than or equal to about10%, less than or equal to about 8%, less than or equal to about 6%,less than or equal to about 5%, less than or equal to about 4%, lessthan or equal to about 3%, less than or equal to about 2%, less than orequal to about 1.5%, less than or equal to about 1%, less than or equalto about 0.8%, less than or equal to about 0.6%, less than or equal toabout 0.5%, less than or equal to about 0.4%, less than or equal toabout 0.3%, less than or equal to about 0.2%, less than or equal toabout 0.1% less than or equal to about 0.05%, less than or equal toabout 0.02%, less than or equal to about 0.01%, less than or equal toabout 0.003%, or less than or equal to about 0.001%, w/v. Combinationsof the above-referenced ranges are also possible (e.g., at least about0.5% and less than or equal to 5% w/v). Other ranges are also possible.

In certain embodiments, a divalent metal salt of bromfenac (e.g.,bromfenac calcium) is present in a composition and/or formulationdescribed herein at about 0.09% w/v or greater. In certain embodiments,the divalent metal salt of bromfenac (e.g., bromfenac calcium) ispresent in a composition and/or formulation described herein at about0.5% w/v or greater. In certain embodiments, diclofenac FA or ketorolacFA, or a metal salt thereof (e.g., divalent or trivalent metal salt), ispresent in a composition and/or formulation described herein at about0.5% w/v or greater.

In some embodiments, the compositions and/or formulations including MPPsof divalent metal salts of bromfenac or other pharmaceutical agentsdescribed herein may have a pH that is not irritating to the eye, suchas a mildly basic pH (e.g., pH 8), physiological pH (i.e., about pH7.4), a substantially neutral pH (e.g., about pH 7), a mildly acidic pH(e.g., about pH 5-6), or ranges thereof (e.g., about pH 5-7, or 6-7). Atthese pHs, the MPPs, compositions, and/or formulations may be chemicallyand colloidally stable and may achieve therapeutically and/orprophylactically effective drug levels for a longer duration in oculartissues compared to certain marketed formulations. The benefitsdescribed herein may further lead to a lower required dose for improvedsafety of this treatment and/or enhanced topical delivery for treatingconditions in the middle and back of the eye compared to certainmarketed formulations.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage blephartis in a subject. Blepharitis is a common eye disorderaffecting people of all ages, and is an inflammation of the eyelid thatresults in itchy, red, irritated eyelids and dandruff-like scales on theeyelashes. The disorder can be caused by bacteria or a skin condition(e.g., dandruff or acne rosacea). Blepharitis symptoms include dry eyes,a gritty sensation or burning in the eyes, itching, swollen and redeyelids, excessive tearing, and crusty eyelids. In more severe cases,blepharitis can cause blurry vision and inflammation of other eyetissue, particularly the cornea.

In one embodiment, the methods, particles, compositions, and/orformulations comprising loteprednol etabonate described herein may beused to treat, diagnose, prevent, or manage blephartis in a subject.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage glaucoma in a subject. Glaucoma is an eye disease in which theoptic nerve is damaged in a characteristic pattern. This can permanentlydamage vision in the affected eye and lead to blindness if leftuntreated. It is normally associated with increased fluid pressure inthe eye (aqueous humour). The term ocular hypertension is used forpeople with consistently raised IOP without any associated optic nervedamage. Conversely, the term normal tension or low tension glaucoma isused for those with optic nerve damage and associated visual field lossbut normal or low IOP.

The nerve damage involves loss of retinal ganglion cells in acharacteristic pattern. There are many different subtypes of glaucoma,but they can all be considered to be a type of optic neuropathy. Raisedintraocular pressure (e.g., above 21 mmHg or 2.8 kPa) is the mostimportant and only modifiable risk factor for glaucoma. However, somemay have high eye pressure for years and never develop damage, whileothers can develop nerve damage at a relatively low pressure. Untreatedglaucoma can lead to permanent damage of the optic nerve and resultantvisual field loss, which over time can progress to blindness.

Current treatment of glaucoma may include the use of prostaglandinanalogs which increase aqueous humor outflow (e.g., Xalatan® (0.005%latanoprost), Lumigan® (0.03% and 0.01% bimatoprost), and Travatan Z®(0.004% travoprost)); beta-blockers which decreases aqueous humorproduction (e.g., Timoptic® (0.5% and 0.25% timolol)); alpha agonistswhich both decrease aqueous humor production and increase outflow (e.g.,Alphagan® (0.1% and 0.15% brimonidine tartrate)); carbonic anhydraseinhibitors which decreases aqueous humor production (e.g., Trusopt® (2%dorzolamide)); and cholinergics (miotic) which increase conventionaloutflow (e.g., Isopto® (1%, 2%, and 4% pilocarpine)). In someembodiments, the particles, formulations, and compositions describedherein may address the issues described above by facilitating effectivedelivery of pharmaceutical agents to the appropriate tissues andavoiding or minimizing clearance of the pharmaceutical agent. Forexample, the particles, compositions, and/or formulations describedherein may include one or more of these or other prostaglandin analogs,beta-blockers, alpha agonists, carbonic anhydrase inhibitors andcholinergics, and may include a coating described herein to facilitatepenetration of the particle through mucus and allow effective deliveryof the pharmaceutical agent.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage uveitis in a subject. Uveitis is inflammation of the uvea, thevascular layer of the eye sandwiched between the retina and the white ofthe eye (sclera). The uvea extends toward the front of the eye andconsists of the iris, choroid layer and ciliary body. The most commontype of uveitis is an inflammation of the iris called iritis (anterioruveitis). Uveitis may also occur at the posterior segment of the eye(e.g., at the choroid). Inflammation of the uvea can be recurring andcan cause serious problems such as blindness if left untreated (accountsfor 10% of blindness globally). Early diagnosis and treatment areimportant to prevent the complications of uveitis.

Current treatment of uveitis includes eye drops (e.g., TobraDex® (0.1%dexamethasone/0.3% tobramycin) and Zylet® (0.5% loteprednoletabonate/0.3% tobramycin)); intravitreal injections of “gel suspension”in sodium hyaluronate (e.g., Trivaris® (8% triamcinolone acetonide));intravitreal injection of “aqueous suspension” in carboxymethylcelluloseand Tween 80 (e.g., Triesence® (4% triamcinolone acetonide)); andimplants (e.g., Retisert® (0.59 mg fluocinolone acetonide) and Ozurdex®(0.7 mg dexamethasone)). Oral steroids and NSAIDS are also employed.Challenges in developing new treatment of uveitis include non-invasivedelivery for posterior uveitis, clearance due to high vascularization ofthe uvea, side effects of long term steroid use such as increased IOPand cataracts. In some embodiments, the particles, compositions, and/orformulations described herein may include one or more of these drugs,which may be administered topically to a subject.

In some embodiments, the methods, particles, compositions, and/orformulations described herein may be used to treat, diagnose, prevent,or manage age-related macular degeneration (AMD) in a subject. AMD is amedical condition which usually affects older adults and results in aloss of vision in the center of the visual field (the macula) because ofdamage to the retina. It occurs in “dry” and “wet” forms. It is a majorcause of blindness and visual impairment in older adults (>50 years).Macular degeneration can make it difficult or impossible to read orrecognize faces, although enough peripheral vision remains to allowother activities of daily life. The macula is the central area of theretina, which provides the most detailed central vision. In the dry(nonexudative) form, cellular debris called drusen accumulate betweenthe retina and the choroid, and the retina can become detached. In thewet (exudative) form, which is more severe, blood vessels grow up fromthe choroid behind the retina, and the retina can also become detached.It can be treated with laser coagulation, and with medication that stopsand sometimes reverses the growth of blood vessels. Although somemacular dystrophies affecting younger individuals are sometimes referredto as macular degeneration, the term generally refers to age-relatedmacular degeneration (AMD or ARMD).

Age-related macular degeneration begins with characteristic yellowdeposits (drusen) in the macula, between the retinal pigment epitheliumand the underlying choroid. Most people with these early changes(referred to as age-related maculopathy) have good vision. People withdrusen can go on to develop advanced AMD. The risk is considerablyhigher when the drusen are large and numerous and associated withdisturbance in the pigmented cell layer under the macula. Recentresearch suggests that large and soft drusen are related to elevatedcholesterol deposits and may respond to cholesterol-lowering agents.

Potential treatment of AMD includes used of pharmaceutical agents suchas verteporfin (e.g., Chlorin®, Visudyne®), thalidomide (e.g.,Ambiodry®, Synovir®, Thalomid®), talaporfin sodium (e.g., Aptocine®,Laserphyrin®, Litx®), ranibizumab (e.g., Lucentis®), pegaptaniboctasodium (e.g., Macugen®, Macuverse®), isopropyl unoprostone (e.g.,Ocuseva®, Rescula®), interferon beta (e.g., Feron®), fluocinoloneacetonide (e.g., Envision TD®, Retisert®), everolimus (e.g., Afinitor®,Certican®, Votubia®, Zortress®), eculizumab (e.g., Solaris®, Soliris®),dexamethasone (e.g., Osurdex®, Ozurdex®, Posurdex®, Surodex®),canakinumab (e.g., Ilaris®), bromfenac (Bromday®), ophthalmic (e.g.,Bronac®, Bronuck®, Xibrom®, Yellox®), brimonidine (e.g., Alphagan®,Bromoxidine®, Enidin®), anecortave acetate (e.g., Retaane®, Edex®,Prostavasin®, Rigidur®, Vasoprost®, Viridal®), aflibercept ophthalmicsolution (e.g., Eyelea®, Eylea®, VEGF-Trap-Eye®), ocriplasmin (e.g.,Iluvien®, Medidur®, Medidur FA®), sirolimus (e.g., Perceiva®), NT-501,KH-902, fosbretabulin tromethamine (e.g., Zybrestat®), AL-8309,aganirsen (e.g., Norvess®), volociximab (e.g., Opthotec®), triamcinolone(e.g., Icon Bioscience), TRC-105, Burixafor (e.g., TG-0054), TB-403(e.g., R-7334), squalamine (e.g., Evizon®), SB-623, S-646240,RTP-801i-14 (e.g., PF-4523655), RG-7417 (e.g., FCFD-45145), AL-78898A(e.g., POT-4), PG-11047 (e.g., CGC-11047), pazopanib hydrochloride,sonepcizumab (e.g., Asonep®, Sphingomab®), padeliporfin (e.g., Stakel®),OT-551, ontecizumab, NOX-A12, hCNS-SC, Neu-2000, NAFB001, MA09-hRPE,LFG-316, iCo-007 (e.g., ISIS-13650), hl-con1, GSK-933776A, GS-6624(e.g., AB-0024), ESBA-1008, epitalon, E-10030 (e.g., ARC-127),dalantercept, MP-0112, CNTO-2476, CERE-120, AAV-NTN, CCX-168,Brimonidine-DDS, bevasiranib sodium (e.g., Cand5), bertilimumab,AVA-101, ALG-1001, AL-39324, AGN-150998, ACU-4429, A6 (e.g., Paralit®),TT-30, sFLT-01 gene therapy, RetinoStat®, PRS-050 (e.g., Angiocal®),PF-4382923, Palomid-529, MC-1101, GW-824575, Dz13 (e.g., TRC-093), D93,CDX-1135 (e.g., TP10), ATL-1103, ARC-1905, XV-615, wet-AMD antibodies(e.g., pSivida), VEGF/rGel, VAR-10200, VAL-566-620-MULTI, TKI, TK-001,STP-601, dry AMD stem cell therapy (e.g., EyeCyte), OpRegen, SMT-D004,SAR-397769, RTU-007, RST-001, RGNX-004, RFE-007-CAI, retinal degeneratonprogramme (e.g., Orphagen), retinal cells (e.g., ISCO), ReN003, PRM-167,ProDex, Photoswitches (e.g., Photoswitch Biosciences), Parkinson'stherapy, OMS-721, 00-10X, NV. AT.08, NT-503, NAFB002, NADPH oxidaseinhibitors (e.g., Alimera Sciences), MC-2002, lycium anti-angiogenicproteoglycan, IXSVEGF, integrin inhibitors, GW-771806, GBS-007, Eos-013,EC-400, dry-AMD therapy (e.g., Neuron Systems), CGEN-25017, CERE-140,AP-202, AMD therapy (e.g., Valens Therapeutics), AMD therapy (e.g.,Amarna Therapeutics), AMD RNAi therapy (e.g., RXi), ALK-001, AMD therapy(e.g., Aciont), AC-301, 4-IPP, zinc-monocysteine complexes (e.g.,Adeona), vatalanib, TG-100-344, prinomastat, PMX-53, Neovastat,mecamylamine, JSM-6427, JPE-1375, CereCRIB, BA-285, ATX-S10, AG-13958,verteporfin/alphavβ3 conjugate, VEGF/rGel, VEGF-saporin, VEGF-R2antagonist (e.g., Allostera), VEGF inhibitors (e.g., Santen), VEGFantagonists (e.g., Ark), Vangiolux®, Triphenylmethanes (e.g., Alimera),TG-100-801, TG-100-572, TA-106, T2-TrpRS, SU-0879, stem cell therapy(e.g., Pfizer and UCL), SOD mimetics (e.g., Inotek), SHEF-1, rostaporfin(e.g., Photrex®, Purlytin®, SnET2), RNA interference (e.g., Idera andMerck), rhCFHp (e.g., Optherion), retino-NPY, retinitis pigmentosatherapy (e.g., Mimetogen), AMD gene therapy (e.g., Novartis), retinalgene therapy (e.g., Genzyme), AMD gene therapy (e.g., Copernicus),retinal dystrophy ther (e.g., Fovea and Genzyme), Ramot project No.K-734B, PRS-055, porcine RPE cells (e.g., GenVec), PMI-002, PLG-101(e.g., BiCentis®), PJ-34, PI3K conjugates (e.g., Semafore), PhotoPoint,Pharmaprojects No. 6526, pegaptanib sodium (e.g., SurModics®), PEDF ZFPTF, PEDF gene therapy (e.g., GenVec), PDS-1.0, PAN-90806, Opt-21,OPK-HVB-010, OPK-HVB-004, Ophthalmologicals (e.g., Cell NetwoRx),ophthalmic compounds (e.g., AstraZenca and Alcon), OcuXan, NTC-200,NT-502, NOVA-21012, Neurosolve®, neuroprotective (e.g., BDSI), MEDI-548,MCT-355, McEye®, LentiVue®, LYN-002, LX-213, lutetium texaphyrin (e.g.,Antrin®), LG-339 inhibitors (e.g., Lexicon), KDR kinase inhibitors(e.g., Merck), ISV-616, INDUS-815C, ICAM-1 aptamer (e.g., Eyetech),hedgehog antagonists (e.g., Opthalmo), GTx-822, GS-102, GranzymeB/VEGF®, gene therapy (e.g., EyeGate), GCS-100 analogue programme,FOV-RD-27, fibroblast growth factor (e.g., Ramot), fenretinide, F-200(e.g., Eos-200-F), Panzem SR®, ETX-6991, ETX-6201, EG-3306, Dz-13,disulfiram (e.g., ORA-102), Diclofenac (e.g., Ophthalmopharma), ACU-02,CLT-010, CLT-009, CLT-008, CLT-007, CLT-006, CLT-005, CLT-004, CLT-003(e.g., Chirovis®), CLT-001, Cethrin® (e.g., BA-210), celecoxib, CD91antagonist (e.g., Ophthalmophar), CB-42, BNC-4, bestrophin, batimastat,BA-1049, AVT-2, AVT-1, atu012, Ape1 programme (e.g., Apex-2), anti-VEGF(e.g., Gryphon), AMD ZFPs (e.g., ToolGen), AMD therapy (e.g.,Optherion), AMD therapy (e.g., ItherX), dry AMD therapy (e.g., Opko),AMD therapy (e.g., CSL), AMD therapies (e.g., Pharmacopeia andAllergan), AMD therapeutic protein (e.g., ItherX), AMD RNAi therapy(e.g., BioMolecular Therapeutics), AM-1101, ALN-VEG01, AK-1003,AGN-211745, ACU-XSP-001 (e.g., Excellair®), ACU-HTR-028, ACU-HHY-011,ACT-MD (e.g., NewNeural), ABCA4 modulators (e.g., Active Pass), A36(e.g., Angstrom), 267268 (e.g., SB-267268), bevacizumab (e.g.,Avastin®), aflibercept (e.g., Eylea®), 131-I-TM-601, vandetanib (e.g.,Caprelsa®, Zactima®, Zictifa®), sunitinib malate (e.g., Sutene®,Sutent®), sorafenib (e.g., Nexavar®), pazopanib (e.g., Armala®,Patorma®, Votrient®), axitinib (e.g., Inlyta®), tivozanib, XL-647,RAF-265, pegdinetanib (e.g., Angiocept®), pazopanib, MGCD-265,icrucumab, foretinib, ENMD-2076, BMS-690514, regorafenib, ramucirumab,plitidepsin (e.g., Aplidin®), orantinib, nintedanib (e.g., Vargateff),motesanib, midostaurin, linifanib, telatinib, lenvatinib, elpamotide,dovitinib, cediranib (e.g., Recentin®), JI-101, cabozantinib, brivanib,apatinib, Angiozyme®, X-82, SSR-106462, rebastinib, PF-337210, IMC-3C5,CYC116, AL-3818, VEGFR2 inhibitor (e.g., AB Science), VEGF/rGel (e.g.,Clayton Biotechnologies), TLK-60596, TLK-60404, R84 antibody (e.g.,Peregrine), MG-516, FLT4 kinase inhibitors (e.g., Sareum), flt-4 kinaseinhibitors, Sareum, DCC-2618, CH-330331, XL-999, XL-820, vatalanib,SU-14813, semaxanib, KRN-633, CEP-7055, CEP-5214, ZK-CDK, ZK-261991,YM-359445, YM-231146, VEGFR2 kinase inhibitors (e.g., Takeda), VEGFR-2kinase inhibitors (e.g., Hanmi), VEGFR-2 antagonist (e.g., Affymax),VEGF/rGel (e.g., Targa), VEGF-TK inhibitors (e.g., AstraZeneca),tyrosine kinase inhibitors (e.g., Abbott), tyrosine kinase inhibitors(e.g., Abbott), Tie-2 kinase inhibitors (e.g., GSK), SU-0879, SP-5.2,sorafenib bead (e.g., Nexavar® bead), SAR-131675, Ro-4383596, R-1530,Pharmaprojects No. 6059, OSI-930, OSI-817, OSI-632, MED-A300,L-000021649, KM-2550, kinase inhibitors (e.g., MethylGene), kinaseinhibitors (e.g., Amgen), Ki-8751, KDR kinase inhibitors (e.g.,Celltech), KDR kinase inhibitors (e.g., Merck), KDR kinase inhibitors(e.g., Amgen), KDR inhibitors (e.g., Abbott), KDR inhibitor (e.g.,LGLS), JNJ-17029259, IMC-1C11, Flt 3/4 anticancer (e.g., Sentinel),EG-3306, DP-2514, DCC-2157, CDP-791, CB-173, c-kit inhibitors (e.g.,Deciphera), BIW-8556, anticancers (e.g., Bracco and Dyax), anti-Flt-1MAbs (e.g., ImClone), AGN-211745, AEE-788, and AB-434.

Laser therapy is also available for wet AMD. Small molecule anti-VEGFtherapies are being investigated, but none are currently approved.Challenges in developing new treatment of AMD include identifyingtreatments, delivery to the macula, side effects and patient compliancewith intravitreal injection.

In addition to other bodily conditions described herein, in someembodiments, the methods, particles, compositions, and/or formulationsdescribed herein may be used to treat, diagnose, prevent, or managemacular edema (e.g., cystoid macular edema (CME) or (diabetic macularedema (DME)) in a subject. CME is a disorder which affects the centralretina or macula of the eye. When this condition is present, multiplecyst-like (cystoid) areas of fluid appear in the macula and causeretinal swelling or edema. CME may accompany a variety of diseases suchas retinal vein occlusion, uveitis, and/or diabetes. CME commonly occursafter cataract surgery.

Currently treatment of CME includes administration of an NSAID (such asbromfenac (e.g., Bromday®)). The NSAID may be co-administered topicallyor intravitreally with a corticosteroid. Severe and persistent cases ofCME are usually treated by intravitreal injection of corticosteroids,which is an invasive and costly procedure.

DME occurs when blood vessels in the retina of patients with diabetesbegin to leak into the macula, the part of the eye responsible fordetailed central vision. These leaks cause the macula to thicken andswell, progressively distorting acute vision. While the swelling may notlead to blindness, the effect can cause a severe loss in central vision.

Currently treatment of DME includes Lucentis® (ranibizumab) injectionsthat are inconvenient and invasive. Another treatment of DME is laserphotocoagulation. Laser photocoagulation is a retinal procedure in whicha laser is used to cauterize leaky blood vessels or to apply a patternof burns to reduce edema. This procedure has undesirable side effectsincluding partial loss of peripheral and night vision.

Due to the drawbacks described above, there is a need for an improvedformulation for the treatment and/or prevention of macular edema (e.g.,CME or DME). The particles, compositions, and/or formulations describedherein including an NSAID (e.g., bromfenac calcium) are stable at a pHsuitable for topical administration to the eye and may address theissues described above with respect to current methods of treatment formacular edema. (See, for example, Examples 27-28). Likewise, it isexpected that the particles, compositions, and/or formulations describedherein including other pharmaceutical agents (e.g. loteprednoletabonate) may also be effective in the treatment, prevention and/ormanagement of macular edema (e.g., CME or DME)

The particles, compositions, and/or formulations described herein may bedelivered into the eye by a variety of routes including, withoutlimitation, orally in any acceptable form (e.g., tablet, liquid,capsule, powder, and the like); topically in any acceptable form (e.g.,patch, eye drops, creams, gels, nebulization, punctal plug, drug elutingcontact, iontophoresis, and ointments); by injection in any acceptableform (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous,parenteral, and epidural); and by implant or the use of reservoirs(e.g., subcutaneous pump, intrathecal pump, suppository, biodegradabledelivery system, non-biodegradable delivery system and other implantedextended or slow release device or formulation).

Partly because the administration of particles, compositions, and/orformulations into the eye by injections is invasive (causing discomfortfor the patient and can also lead to complications that are even moreserious than the disease being treated) and oral doses often lead to lowdistribution of the particles, compositions, and/or formulations intothe eye, topical delivery may be preferred in some embodiments. The keybenefits of topical delivery include non-invasive character, localizedaction with reduced systemic exposure, relative patient comfort, andease of administration.

Compliance is an issue which stems from a wide variety of factors, frompatients' difficulty remembering to take drops, to trouble in physicallyadministering drops, to unpleasant side effects. Other issues includerapid clearance of drug and systemic exposure.

As described herein, in some embodiments, the particles, compositions,and/or formulations may be administered topically to an eye of thesubject, and a pharmaceutical agent may be delivered to a posterior partof the eye (e.g. to retina, choroid, vitreous, and optic nerve). Theparticles, compositions, and/or formulations may be used to treat,diagnose, prevent, or manage a disorder such as age-related maculardegeneration, diabetic retinopathy, retinal venous occlusions, retinalarterial occlusion, macular edema, postoperative inflammation, uveitisretinitis, proliferative vitreoretinopathy and glaucoma.

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the imaging of the eye. In certainembodiments, the particles, compositions, and methods described hereinare useful in the diagnosis of an ocular condition.

In some embodiments, ophthalmic delivery of a pharmaceutical compositiondescribed herein includes delivery to an ocular surface, to the lacrimalglands or lacrimal drainage system, to the eyelids, to the anteriorsegment of the eye, to the posterior segment of the eye, and/or to theperiocular space. In certain embodiments, a pharmaceutical compositiondescribed herein can be delivered to the cornea, iris/ciliary body,aqueous humor, vitreous humor, retina, choroid and/or sclera. Thetherapeutic effect of delivering a pharmaceutical composition describedherein may be improved compared to the effect of delivering of particlesthat are not identified herein as mucus penetrating.

In some embodiments, the pharmaceutical agent that is delivered into theeye by the particles, compositions, and/or methods described herein maybe a corticosteroid. In certain embodiments, the pharmaceutical agent isloteprednol etabonate. In certain embodiments, the pharmaceutical agentincludes one or more of hydrocortisone, cortisone, tixocortol,prednisolone, methylprednisolone, prednisone, triamcinolone, mometasone,amcinonide, budesonide, desonide, fluocinonide, fluocinolone,halcinonide, betamethasone, dexamethasone, fluocortolone,hydrocortisone, aclometasone, prednicarbate, clobetasone, clobetasol,fluprednidene, glucocorticoid, mineralocorticoid, aldosterone,deoxycorticosterone, fludrocortisone, halobetasol, diflorasone,desoximetasone, fluticasone, flurandrenolide, alclometasone,diflucortolone, flunisolide, and beclomethasone.

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the delivery of a corticosteroid, such asone described above, into the eye for the treatment of inflammation ofthe eye. In certain embodiments, the particles, compositions, andmethods are useful in the delivery of a corticosteroid into the eye forthe treatment of macular degeneration, macular edema, other retinaldisorders, or other conditions described herein.

In some embodiments, the pharmaceutical agent that is delivered into theeye by the particles, compositions, and methods described herein may bea non-steroidal anti-inflammatory drug (NSAID). In certain embodiments,the pharmaceutical agent is a divalent metal salt of bromfenac (e.g.,bromfenac calcium). In certain embodiments, the pharmaceutical agent isdiclofenac (e.g., diclofenac free acid or a divalent or trivalent metalsalt thereof). In certain embodiments, the pharmaceutical agent isketorolac (e.g., ketorolac free acid or a divalent or trivalent metalsalt thereof). In certain embodiments, the pharmaceutical agent is asalicylate (e.g., aspirin (acetylsalicylic acid), diflunisal, orsalsalate). In certain embodiments, the pharmaceutical agent is apropionic acid derivative (e.g., ibuprofen, naproxen, fenoprofen,ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, and loxoprofen). Incertain embodiments, the pharmaceutical agent is an acetic acidderivative (e.g., indomethacin, sulindac, etodolac, ketorolac,diclofenac, and nabumetone). In certain embodiments, the pharmaceuticalagent is an enolic acid (oxicam) derivative (e.g., piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, and isoxicam). In certain embodiments,the pharmaceutical agent is a fenamic acid derivative (fenamate) (e.g.,mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamicacid). In certain embodiments, the pharmaceutical agent is acyclooxygenase (cox) inhibitor, such as a cox-1 or cox-2 inhibitor(e.g., bromfenac calcium). In certain embodiments, the pharmaceuticalagent is a selective cox-2 inhibitor (coxib) (e.g., celecoxib,rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, andfirocoxib). In certain embodiments, the pharmaceutical agent is asulphonanilide (e.g., nimesulide). In certain embodiments, thepharmaceutical agent is licofelone.

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the delivery of an NSAID, such as onedescribed above, into the eye for the treatment of inflammation of theeye or other conditions described herein. In some embodiments, thepharmaceutical agent that is delivered into the eye by the particles,compositions, and methods described herein may be an angiogenesisinhibitor. In certain embodiments, the pharmaceutical agent is anendogenous angiogenesis inhibitor (e.g., VEGFR-1 (e.g., pazopanib(Votrient®), cediranib (Recentin®), tivozanib (AV-951), axitinib(Inlyta®), semaxanib), HER2 (lapatinib (Tykerb®, Tyverb®), linifanib(ABT-869), MGCD-265, and KRN-633), VEGFR-2 (e.g., regorafenib (BAY73-4506), telatinib (BAY 57-9352), vatalanib (PTK787, PTK/ZK), MGCD-265,OSI-930, and KRN-633), NRP-1, angiopoietin 2, TSP-1, TSP-2, angiostatin,endostatin, vasostatin, calreticulin, platelet factor-4, TIMP, CDAI,Meth-1, Meth-2, IFN-α, IFN-β, IFN-γ, CXCL10, IL-4, IL-12, IL-18,prothrombin (kringle domain-2), antithrombin III fragment, prolactin,VEGI, SPARC, osteopontin, maspin, canstatin, a proliferin-relatedprotein, sorafenib (Nexavar®)), and restin). In certain embodiments, thepharmaceutical agent is an exogenous angiogenesis inhibitor (e.g.,bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN-α,IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFRantagonist, an angiostatic steroid+heparin, a cartilage-derivedangiogenesis inhibitory factor, a matrix metalloproteinase inhibitor,angiostatin, endostatin, 2-methoxyestradiol, tecogalan,tetrathiomolybdate, thalidomide, thrombospondin, prolactin, a α_(V)β₃inhibitor, linomide, and tasquinimod).

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the delivery of an angiogenesisinhibitor, such as those described above, into the eye for the treatmentof macular degeneration, other retinal disorders, or other conditionsdescribed herein. In some embodiments, the pharmaceutical agent that isdelivered into the eye by the particles, compositions, and methodsdescribed herein may be a prostaglandin analog. In certain embodiments,the pharmaceutical agent is latanoprost, travoprost, unoprostone, orbimatoprost.

In some embodiments, the pharmaceutical agent in a particle, compositionand/or formulation described herein is an RTK inhibitor. In certainembodiments, the pharmaceutical agent is sorafenib. For example, asdescribed in more detail in Examples 21, 25, and 29, administration ofparticles of sorafenib that included certain surface-altering agentsdescribed herein resulted in markedly higher sorafenib levels in variousocular tissues (e.g., tissues at the back of the eye) in rabbits,compared to an equivalent dose of particles of sorafenib that do notinclude a suitable surface-altering agent.

In certain embodiments, the pharmaceutical agent in a particle,composition and/or formulation described herein is linifanib. Forexample, as described in more detail in the Example 29, administrationof MPPs containing linifanib enhanced the exposure of linifanib at theback of the eye of rabbits.

In certain embodiments, the pharmaceutical agent in a particle,composition and/or formulation described herein is MGCD-265. Forexample, as described in more detail in the Example 30, administrationof MPPs containing MGCD-265 resulted in therapeutically-relevant levelsof MGCD-265 at the back of the eye of rabbits.

In certain embodiments, the pharmaceutical agent in a particle,composition and/or formulation described herein is pazopanib. Forexample, as described in more detail in the Example 30, administrationof MPPs containing pazopanib generated therapeutically relevant levelsof pazopanib at the back of the eye of rabbits.

In certain embodiments, the pharmaceutical agent in a particle,composition and/or formulation described herein is cediranib. Forexample, as described in more detail in the Example 31, a single topicaladministration of cediranib-MPPs produced therapeutically relevantcediranib levels at the back of the eye of rabbits for 24 hours.

In certain embodiments, the pharmaceutical agent in a particle,composition and/or formulation described herein is axitinib. Forexample, as described in more detail in the Examples 32 and 33, a singletopical administration of axitinib-MPP resulted in therapeuticallyrelevant axitinib levels at the back of the eye of rabbits for 24 hours,and axitinib-MPP reduced vascular leakage in a rabbit VEGF (vascularendothelial growth factor receptor)-challenge model.

The results described above and herein suggest that the particles,compositions, and/or formulations described herein may be administeredtopically to achieve and sustain therapeutic effect in treating AMD,other retinal disorders, or other conditions described herein, comparedto certain marketed formulations such as those which must be injectedinto the eye.

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the delivery of a prostaglandin analog,such as one described above, into the eye for the treatment of glaucomaor other condition described herein.

In some embodiments, the pharmaceutical agent that is delivered into theeye by the particles, compositions, and methods described herein may bea beta blocker. In certain embodiments, the pharmaceutical agent is anon-selective beta blocker (e.g., alprenolol, bucindolol, carteolol,carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol,propranolol, sotalol, timolol, and eucommia bark). In certainembodiments, the pharmaceutical agent is a β₁-selective blocker (e.g.,acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol,metoprolol, and nebivolol). In certain embodiments, the pharmaceuticalagent is a β₂-selective blocker (e.g., butaxamine and ICI-118,551). Incertain embodiments, the pharmaceutical agent is a β₃-selective blocker(e.g., SR 59230A).

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the delivery of a beta blocker, such asone described above, into the eye for the treatment of glaucoma or othercondition described herein.

In certain embodiments, the pharmaceutical agent that is delivered intothe eye by the particles, compositions, and methods of the presentinvention may be a carbonic anhydrase inhibitor. In certain embodiments,the pharmaceutical agent is acetazolamide, brinzolamide, dorzolamide,dorzolamide and timolol, or methazolamide.

In certain embodiments, the particles, compositions, and methodsdescribed herein are useful in the delivery of a carbonic anhydraseinhibitor, such as those described above, into the eye for the treatmentof glaucoma or other conditions described herein. As described herein,in some embodiments, the particles, compositions, and/or formulationsdescribed herein can improve or increase ocular bioavailability, definedas the area under the curve (AUC) of drug concentration in an oculartissue of interest against time after administration, of apharmaceutical agent that is administered topically to an eye of asubject compared to certain existing particles, compositions, and/orformulations. In some embodiments, the ocular bioavailability of thepharmaceutical agent may increase due to, at least in part, a coating oncore particles comprising the pharmaceutical agent that renders theparticles mucus penetrating, compared to particles of the pharmaceuticalagent of similar size as the coated particle in question, but which doesnot include the coating.

In some embodiments, the particles, compositions, and/or formulationsdescribed herein increase the ocular bioavailability of a pharmaceuticalagent by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 150%, at least about 200%, at least about 5 fold, at leastabout 10 fold, at least about 20 fold, at least about 50 fold, at leastabout 100 fold, at least about 500 fold, or at least about 1000 fold. Incertain the particles, compositions, and/or formulations describedherein increase the ocular bioavailability of a pharmaceutical agent byless than or equal to about 1000 fold, less than or equal to about 500fold, less than or equal to about 100 fold, less than or equal to about50 fold, less than or equal to about 20 fold, less than or equal toabout 10 fold, less than or equal to about 5 fold, less than or equal toabout 200%, less than or equal to about 150%, less than or equal toabout 100%, less than or equal to about 90%, less than or equal to about80%, less than or equal to about 70%, less than or equal to about 60%,less than or equal to about 50%, less than or equal to about 40%, lessthan or equal to about 30%, less than or equal to about 20%, or lessthan or equal to about 10%. Combinations of the above-referenced rangesare also possible (e.g., an increase of at least about 10% and less thanor equal to about 10 fold). Other ranges are also possible. In someinstances, the AUC of a pharmaceutical agent increases at a tissueand/or fluid in the front of the eye. In other instances, the AUC of apharmaceutical agent increases at a tissue and/or fluid in the back ofthe eye.

In general, an increase in ocular bioavailability may be calculated bytaking the difference in the AUC measured in an ocular tissue ofinterest (e.g., in aqueous humor) between those of a test compositionand a control composition, and dividing the difference by thebioavailability of the control composition. A test composition mayinclude particles comprising a pharmaceutical agent, and the particlesmay be characterized as being mucus penetrating (e.g., having a relativevelocity in mucus of greater than about 0.5, or another other relativevelocity described herein). A control composition may include particlescomprising the same pharmaceutical agent as that present in the testcomposition, the particles having a substantially similar size as thoseof the test composition, but which are not mucus penetrating (e.g.,having a relative velocity in mucus of less than or equal to about 0.5,or another other relative velocity described herein).

Ocular bioavailability of a pharmaceutical agent may be measured in anappropriate animal model (e.g. in a New Zealand white rabbit model). Theconcentration of a pharmaceutical agent and, when appropriate, itsmetabolite(s), in appropriate ocular tissues or fluids is measured as afunction of time after administration.

Other methods of measuring ocular bioavailability of a pharmaceuticalagent are possible.

As described herein, in some embodiments, the concentration of apharmaceutical agent in an ocular tissue and/or fluid may be increasedwhen the pharmaceutical agent is delivered (e.g., via topicaladministration to the eye) using the particles, compositions, and/orformulations described herein compared to when the pharmaceutical agentis delivered using certain existing particles, compositions, and/orformulations that contain the same the pharmaceutical agent (or comparedto the delivery of the same pharmaceutical agent (e.g., of similar size)as the coated particle in question, but which does not include thecoating). In certain embodiments, a dose of the particles, compositions,and/or formulations is administered, followed by the measurement of theconcentration of the pharmaceutical agent in a tissue and/or fluid ofthe eye. For purposes of comparison, the amount of the pharmaceuticalagent included in the administered dose of the particles, compositions,and/or formulations described herein may be similar or substantiallyequal to the amount of the pharmaceutical agent included in theadministered dose of the existing particles, compositions, and/orformulations. In certain embodiments, the concentration of thepharmaceutical agent in a tissue and/or fluid of the eye is measured ata certain time subsequent to the administration (“time post-dose”) of adose of the particles, compositions, and/or formulations describedherein or of the existing particles, compositions, and/or formulations.In certain embodiments, the time when the concentration is measured isabout 1 min, about 10 min, about 30 min, about 1 h, about 2 h, about 3h, about 4 h, about 5 h, about 6 h, about 7 h, about 8 h, about 9 h,about 10 h, about 11 h, about 12 h, about 18 h, about 24 h, about 36 h,or about 48 h, post-dose.

In some embodiments, the concentration of the pharmaceutical agent in atissue and/or fluid may increase due to, at least in part, a coating oncore particles comprising the pharmaceutical agent that renders theparticles mucus penetrating, compared to particles of the samepharmaceutical agent (e.g., of similar size) as the coated particle inquestion, but which does not include the coating. In some embodiments,the particles, compositions, and/or formulations described hereinincreases the concentration of a pharmaceutical agent in a tissue and/orfluid by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 200%, at least about 300%, at least about 400%, at leastabout 500%, or at least about 10 fold, at least about 20 fold, at leastabout 50 fold, at least about 100 fold, at least about 1000 fold, atleast about 10⁴ fold, at least about 10⁵ fold, or at least about 10⁶fold. In some cases, the particles, compositions, and/or formulationsdescribed herein increases the concentration of a pharmaceutical agentin a tissue and/or fluid by less than or equal to about 10⁶ fold, lessthan or equal to about 10⁵ fold, less than or equal to about 10⁴ fold,1000 fold, less than or equal to about 100 fold, less than or equal toabout 10 fold, less than or equal to about 500%, less than or equal toabout 400%, less than or equal to about 300%, less than or equal toabout 200%, less than or equal to about 100%, less than or equal toabout 90%, less than or equal to about 80%, less than or equal to about70%, less than or equal to about 60%, less than or equal to about 50%,less than or equal to about 40%, less than or equal to about 30%, lessthan or equal to about 20%, or less than or equal to about 10%.Combinations of the above-referenced ranges are also possible (e.g., anincrease of greater than or equal to about 10% and less than or equal toabout 90%). Other ranges are also possible. In some instances, theconcentration of a pharmaceutical agent increases at a tissue and/orfluid in the front of the eye. In other instances, the concentration ofa pharmaceutical agent increases at a tissue and/or fluid in the back ofthe eye.

The ocular concentration of a pharmaceutical agent, and, whenappropriate, its metabolite(s), in appropriate ocular fluids or tissuesmay be measured as a function of time in vivo using an appropriateanimal model. One method of determining the ocular concentration of apharmaceutical agent involves dissecting of the eye to isolate tissuesof interest (e.g., in a animal model comparable to the subject). Theconcentration of the pharmaceutical agent in the tissues of interest isthen determined by HPLC or LC/MS analysis.

In certain embodiments, the period of time between administration of theparticles described herein and obtaining a sample for measurement ofconcentration or AUC is less than about 1 hour, less than or equal toabout 2 hours, less than or equal to about 3 hours, less than or equalto about 4 hours, less than or equal to about 6 hours, less than orequal to about 12 hours, less than or equal to about 36 hours, or lessthan or equal to about 48 hours. In certain embodiments, the period oftime is at least about 1 hour, at least about 2 hours, at least about 3hours, at least about 4 hours, at least about 6 hours, at least about 8hours, at least about 12 hours, at least about 36 hours, or at leastabout 48 hours. Combinations of the above-referenced ranges are alsopossible (e.g., a period of time between consecutive doses of greaterthan or equal to about 3 hours and less than or equal to about 12hours). Other ranges are also possible.

Other methods of measuring the concentration of a pharmaceutical agentin an eye of a subject or an animal model are also possible. In someembodiments, the concentration of a pharmaceutical agent may be measuredin the eye of the subject directly or indirectly (e.g., taking a sampleof fluid, such as vitreous humor, from an eye of the subject).

In general, an increase in concentration of a pharmaceutical agent in anocular site may be calculated by taking the difference in concentrationmeasured between those of a test composition and a control composition,and dividing the difference by the concentration of the controlcomposition. A test composition may include particles comprising apharmaceutical agent, and the particles may be characterized as beingmucus penetrating (e.g., having a relative velocity of greater thanabout 0.5, or another other relative velocity described herein). Acontrol composition may include particles comprising the samepharmaceutical agent as that present in the test composition, theparticles having a substantially similar size as those of the testcomposition, but which are not mucus penetrating (e.g., having arelative velocity of less than about 0.5, or another other relativevelocity described herein).

As described herein, in some embodiments, the particles, compositions,and/or formulations described herein, or a component thereof, is presentin a sufficient amount to increase the bioavailability and/orconcentration of a pharmaceutical agent in an ocular tissue, compared tothe pharmaceutical agent administered to the ocular tissue in theabsence of the particles, compositions, and formulations describedherein, or a component thereof.

The ocular tissue may be an ocular tissue described herein, such as ananterior ocular tissue (e.g., a palpebral conjunctiva, a bulbarconjunctiva, or a cornea). The pharmaceutical agent may be any suitableagent as described herein, such as a corticosteroid (e.g., loteprednoletabonate), an RTK inhibitor (e.g., sorafenib, linifanib, MGCD-265,pazopanib, cediranib, and axitinib), an NSAID (e.g., bromfenac calcium),or a cox inhibitor (e.g., bromfenac calcium). In certain embodiments,the core particle of a formulation comprising a pharmaceutical agent ispresent in a sufficient amount to increase the bioavailability and/orconcentration of the pharmaceutical agent in an ocular tissue. Incertain embodiments, the coating on the core particle of a formulationcomprising a pharmaceutical agent is present in a sufficient amount toincrease the bioavailability and/or concentration of the pharmaceuticalagent in an ocular tissue. In certain embodiments, the coating on thecore particle of a formulation comprising a pharmaceutical agent ispresent in a sufficient amount to increase the concentration of thepharmaceutical agent in an ocular tissue after at least 10 minutes, atleast 20 minutes, at least 30 minutes, at least 1 hour, at least 2hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 9hours, at least 12 hours, at least 18 hours, or at least 24 hours afteradministration of the formulation to the ocular tissue. In certainembodiments, the coating on the core particle of a formulationcomprising a pharmaceutical agent is present in a sufficient amount toincrease the concentration of the pharmaceutical agent in an oculartissue after less than or equal to 24 hours, less than or equal to 18hours, less than or equal to 12 hours, less than or equal to 9 hours,less than or equal to 6 hours, less than or equal to 4 hours, less thanor equal to 3 hours, less than or equal to 2 hours, less than or equalto 1 hour, less than or equal to 30 minutes, less than or equal to 20minutes, or less than or equal to 10 minutes after administration of theformulation to the ocular tissue. Combinations of the above-referencedranges are also possible (e.g., the concentration of the pharmaceuticalagent increases after at least 10 minutes and less than or equal to 2hours). Other ranges are also possible. In certain embodiments, thecoating on the core particle of a formulation comprising apharmaceutical agent is present in a sufficient amount to increase theconcentration of the pharmaceutical agent in an ocular tissue afterabout 30 minutes after administration of the formulation to the oculartissue.

As described herein, in some embodiments, the particles, compositions,and/or formulations described herein can be administered topically to aneye of a subject in various forms of doses. For example, the particles,compositions, and/or formulations described herein may be administeredin a single unit dose or repeatedly administered in a plurality ofsingle unit doses. A unit dose is a discrete amount of the particles,compositions, and/or formulations described herein comprising apredetermined amount of a pharmaceutical agent. In some embodiments,fewer numbers of doses (e.g., ½, ⅓, or ¼ the number doses) are requiredusing the particles described herein having a mucus-penetrating coatingcompared to particles that do not have such a coating.

The exact amount of the particles, compositions, and/or formulationsdescribed herein required to achieve a therapeutically orprophylactically effective amount will vary from subject to subject,depending, for example, on species, age, and general condition of asubject, severity of the side effects or disorder, identity of theparticular compound, mode of administration, and the like. Theparticles, compositions, and/or formulations described herein can bedelivered using repeated administrations where there is a period of timebetween consecutive doses. Repeated administration may be advantageousbecause it may allow the eye to be exposed to a therapeutically orprophylactically effective amount of a pharmaceutical agent for a periodof time that is sufficiently long for the ocular condition to betreated, prevented, or managed. In certain embodiments, the period oftime between consecutive doses is less than or equal to about 1 hour,less than or equal to about 2 hours, less than or equal to about 3hours, less than or equal to about 4 hours, less than or equal to about6 hours, less than or equal to about 12 hours, less than or equal toabout 36 hours, or less than or equal to about 48 hours. In certainembodiments, the period of time between consecutive doses is at leastabout 1 hour, at least about 2 hours, at least about 3 hours, at leastabout 4 hours, at least about 6 hours, at least about 12 hours, at leastabout 36 hours, or at least about 48 hours. Combinations of theabove-referenced ranges are also possible (e.g., a period of timebetween consecutive doses of greater than or equal to about 3 hours andless than or equal to about 12 hours). Other ranges are also possible.

Delivery of the particles, compositions, and/or formulations describedherein to an ocular tissue may result in ophthalmically efficacious druglevels in the ocular tissue for an extended period of time afteradministration (e.g., topical administration or administration by directinjection). An ophthalmically efficacious level of a drug refers to anamount sufficient to elicit the desired biological response of an oculartissue, i.e., treating an ocular disease. As will be appreciated bythose skilled in this art, the ophthalmically efficacious level of adrug may vary depending on such factors as the desired biologicalendpoint, the pharmacokinetics of the drug, the ocular disease beingtreated, the mode of administration, and the age and health of thesubject. In certain embodiments, the ophthalmically efficacious level ofa drug is an amount of the drug, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of theocular condition. The ophthalmically efficacious level of a drug canencompass a level that improves overall therapy, reduces or avoidssymptoms or causes of the ocular condition, or enhances the therapeuticefficacy of another therapeutic agent.

In some embodiments, an ophthalmically efficacious drug level may begauged, at least in part, by the maximum concentration (C_(max)) of thepharmaceutical agent in the ocular tissue after administration. In somecases, delivery of the particles, compositions, and/or formulationscomprising a pharmaceutical agent as described herein to an oculartissue may result in a higher C_(max) of the pharmaceutical agent in theocular tissue after administration, compared to marketed particles,compositions, and formulations at similar doses. In certain embodiments,the C_(max) obtained from an administration of the particles,compositions, and/or formulations described herein is at least about 3%,at least about 10%, at least about 30%, at least about 100%, at leastabout 200%, at least about 300%, at least about 400%, at least about500%, at least about 1000%, or at least about 3000%, higher than theC_(max) obtained from an administration of the marketed particles,compositions, and/or formulations. In certain embodiments, the C_(max)obtained from an administration of the particles, compositions, and/orformulations described herein is less than or equal to about 3000%, lessthan or equal to about 1000%, less than or equal to about 500%, lessthan or equal to about 400%, less than or equal to about 300%, less thanor equal to about 200%, less than or equal to about 100%, less than orequal to about 30%, less than or equal to about 10%, or less than orequal to about 3%, higher than the C_(max) obtained from anadministration of the marketed particles, compositions, and/orformulations. Combinations of the above-referenced ranges are alsopossible (e.g., an increase in C_(max) at least about 30% and less thanor equal to about 500%). Other ranges are also possible.

In some embodiments, the ophthalmically efficacious drug levels aregauged, at least in part, by minimally efficacious concentrations of thedrug, e.g., IC₅₀ or IC₉₀, as known in the art.

In certain embodiments in which ophthalmically efficacious drug levels(or C_(max), IC₅₀, or IC₉₀) are present in the ocular tissue for anextended period of time after administration, the extended period oftime after administration can range from hours to days. In certainembodiments, the extended period of time after administration is atleast 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, atleast 9 hours, at least 12 hours, at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, or atleast 1 week. In certain embodiments, the extended period of time afteradministration is less than or equal to 1 week, less than or equal to 6days, less than or equal to 5 days, less than or equal to 4 days, lessthan or equal to 3 days, less than or equal to 2 days, less than orequal to 1 day, less than or equal to 12 hours, less than or equal to 9hours, less than or equal to 6 hours, less than or equal to 4 hours,less than or equal to 2 hours, less than or equal to 1 hour.Combinations of the above-referenced ranges are also possible (e.g., anextended period of time of at least about 4 hours and less than or equalto about 1 week). Other ranges are also possible.

In certain embodiments, the particles, compositions, and/or formulationsdescribed herein may be at dosage levels sufficient to deliver aneffective amount of a pharmaceutical agent to an eye of a subject toobtain a desired therapeutic or prophylactic effect. In certainembodiments, an effective amount of a pharmaceutical agent that isdelivered to an appropriate eye tissue is at least about 10⁻³ ng/g, atleast about 10⁻² ng/g, at least about 10⁻¹ ng/g, at least about 1 ng/g,at least about 10¹ ng/g, at least about 10² ng/g, at least about 10³ng/g, at least about 10⁴ ng/g, at least about 10⁵ ng/g, or at leastabout 10⁶ ng/g of tissue weight. In certain embodiments, an effectiveamount of a pharmaceutical agent that is delivered to the eye is lessthan or equal to about 10⁶ ng/g, less than or equal to about 10⁵ ng/g,less than or equal to about 10⁴ ng/g, less than or equal to about 10³ng/g, less than or equal to about 10² ng/g, less than or equal to about10¹ ng/g, less than or equal to about 1 ng/g, less than or equal toabout 10⁻¹ ng/g, less than or equal to about 10⁻² ng/g, or less than orequal to about 10⁻³ ng/g of tissue weight. Combinations of theabove-referenced ranges are also possible (e.g., an effective amount ofa pharmaceutical agent of at least about 10⁻² ng/g and less than orequal to about 10³ ng/g of tissue weight). Other ranges are alsopossible. In certain embodiments, the particles, compositions, and/orformulations described herein may be at dosage levels sufficient todeliver an effective amount of a pharmaceutical agent to the back of aneye of a subject to obtain a desired therapeutic or prophylactic effect.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided particles, compositions,and/or formulations to an adult. The amount to be administered to, forexample, a child or an adolescent can be determined by a medicalpractitioner or person skilled in the art and can be lower or the sameas that administered to an adult.

The particles, compositions, and/or formulations described herein may betopically administered by any method, for example, as by drops, powders,ointments, or creams. Other topical administration approaches or formsare also possible.

In certain embodiments, the compositions and/or formulations describedherein are packaged as a ready to use shelf stable suspension. Eye dropformulations are traditionally liquid formulations (solutions orsuspensions) which can be packaged in dropper bottles (which dispense astandard drop volume of liquid) or in individual use droppers (typicallyused for preservative free drops; used once and disposed). Theseformulations are ready to use and can be self-administered. In somecases the bottle should be shaken before use to ensure homogeneity ofthe formulation, but no other preparation may be necessary. This may bethe simplest and most convenient method of ocular delivery. Thecompositions and/or formulations described herein can be packaged in thesame way as traditional eye drop formulations. They can be stored insuspension and may retain the characteristics which allow the particlesto avoid adhesion to mucus.

The pharmaceutical agent may be one of those pharmaceutical agentsdescribed herein. In certain embodiments, the pharmaceutical agent is anNSAID, an RTK inhibitor, a cox inhibitor, a corticosteroid, anangiogenesis inhibitor, a prostaglandin analog, a beta blocker, or acarbonic anhydrase inhibitor. In certain embodiments, the pharmaceuticalagent is loteprednol etabonate. In certain embodiments, thepharmaceutical agent is sorafenib. In certain embodiments, thepharmaceutical agent is linifanib. In certain embodiments, thepharmaceutical agent is MGCD-265. In certain embodiments, thepharmaceutical agent is pazopanib. In certain embodiments, thepharmaceutical agent is cediranib. In certain embodiments, thepharmaceutical agent is axitinib. In certain embodiments, thepharmaceutical agent is a divalent metal salt of bromfenac (e.g.,bromfenac calcium). In certain embodiments, the pharmaceutical agent isbromfenac beryllium, bromfenac magnesium, bromfenac strontium, orbromfenac barium, bromfenac zinc, or bromfenac copper(II). Otherpharmaceutical agents are also possible.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1

The following describes a non-limiting example of a method of formingnon-polymeric solid particles into mucus-penetrating particles. Pyrene,a hydrophobic naturally fluorescent compound, was used as the coreparticle and was prepared by a milling process in the presence ofvarious surface-altering agents. The surface-altering agents formedcoatings around the core particles. Different surface-altering agentswere evaluated to determine effectiveness of the coated particles inpenetrating mucus.

Pyrene was milled in aqueous dispersions in the presence of varioussurface-altering agents to determine whether certain surface-alteringagents can: 1) aid particle size reduction to several hundreds ofnanometers and 2) physically (non-covalently) coat the surface ofgenerated nanoparticles with a mucoinert coating that would minimizeparticle interactions with mucus constituents and prevent mucusadhesion. In these experiments, the surface-altering agents acted as acoating around the core particles, and the resulting particles weretested for their mobility in mucus, although in other embodiments, thesurface-altering agents may be exchanged with other surface-alteringagents that can increase mobility of the particles in mucus. Thesurface-altering agents tested included a variety of polymers,oligomers, and small molecules listed in Table 2, includingpharmaceutically relevant excipients such as poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers(Pluronics®), polyvinylpyrrolidones (Kollidon), and hydroxypropylmethylcellulose (Methocel), etc.

TABLE 2 Surface-altering agents tested with Pyrene as a model compound.Polymeric surface-altering agents Acronym or Trade Grade or MolecularStabilizer Name Weight Chemical Structure Poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide) block copolymers Pluronic ®F127, F108, F68, F87, F38, P123, P105 P103 P65 L121, L101, L81, L44, L31

Polyvinylpyrrolidone PVP Kollidon 17 (9K), Kollidon 25 (26K), Kollindon30 (43K)

PVA-poly (ethylene glycol) graft-copolymer Kollicoat IR

Hydroxypropyl methylcellulose HPMC Methocel E50, Methocel K100

Oligomeric surface-altering agents Tween 20

Tween 80

Solutol HS 15

Triton X100

Tyloxapol

Cremophor RH 40 Small molecule surface-altering agents Span 20

Span 80

Octyl glucoside

Cetytrimethylammonium bromide (CTAB)

Sodium dodecyl sulfate (SDS)

An aqueous dispersion containing pyrene and one of the surface-alteringagents listed above was milled with milling media until particle sizewas reduced below 500 nm. Table 3 lists particle size characteristics ofpyrene particles obtained by milling in the presence of the varioussurface-altering agents. Particle size was measured by dynamic lightscattering. When Pluronics® L101, L81, L44, L31, Span 20, Span 80, orOctyl glucoside were used as surface-altering agents, stablenanosuspensions could not be obtained. Therefore, these surface-alteringagents were excluded from further investigation due to their inabilityto effectively aid particle size reduction.

TABLE 3 Particle size measured by DLS in nanosuspensions obtained bymilling of Pyrene with various surface-altering agents. StabilizerN-Ave. D (nm) Pluronic ® F127 239 Pluronic ® F108 267 Pluronic ® P105303 Pluronic ® P103 319 Pluronic ® P123 348 Pluronic ® L121 418Pluronic ® F68 353 Pluronic ® P65 329 Pluronic ® F87 342 Pluronic ® F38298 Pluronic ® L101 not measurable* Pluronic ® L81 not measurable*Pluronic ® L44 not measurable* Pluronic ® L31 not measurable* PVA 13K314 PVA 31K 220 PVA 85K 236 Kollicoat IR 192 Kollidon 17 (PVP 9K) 163Kollidon 25 (PVP 26K) 210 Kollindon 30 (PVP 43K) 185 Methocel E50 160Methocel K100 216 Tween 20 381 Tween 80 322 Solutol HS 378 Triton X100305 Tyloxapol 234 Cremophor RH40 373 SDS 377 CTAB 354 Span 20 notmeasurable* Span 80 not measurable* Octyl glucoside not measurable**milling with Pluronics ® L101, L81, L44, L31, Span 20, Span 80, Octylglucoside failed to effectively reduce pyrene particle size and producestable nanosuspensions.

The mobility and distribution of pyrene nanoparticles from the producednanosuspensions in human cervicovaginal mucus (CVM) were characterizedusing fluorescence microscopy and multiple particle tracking software.In a typical experiment, ≤1.5 uL of a nanosuspension (diluted ifnecessary to the surfactant concentration of ˜1%) was added to 20 μl offresh CVM along with controls. Conventional nanoparticles (200 nmyellow-green fluorescent carboxylate-modified polystyrene microspheresfrom Invitrogen) were used as a negative control to confirm the barrierproperties of the CVM samples. Red fluorescent polystyrene nanoparticlescovalently coated with PEG 5 kDa were used as a positive control withwell-established MPP behavior. Using a fluorescent microscope equippedwith a CCD camera, 15 s movies were captured at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample (pyrene), negativecontrol, and positive control (natural blue fluorescence of pyreneallowed observing of pyrene nanoparticles separately from the controls).Next, using an advanced image processing software, individualtrajectories of multiple particles were measured over a time-scale of atleast 3.335 s (50 frames). Resulting transport data are presented herein the form of trajectory-mean velocity V_(mean), i.e., velocity of anindividual particle averaged over its trajectory, and ensemble-averagevelocity <V_(mean)>, i.e., V_(mean) averaged over an ensemble ofparticles. To enable easy comparison between different samples andnormalize velocity data with respect to natural variability inpenetrability of CVM samples, relative sample velocity <V_(mean)>_(rel),was determined according to the formula shown in Equation 1.

Prior to quantifying mobility of the produced pyrene nanoparticles,their spatial distribution in the mucus sample was assessed bymicroscopy at low magnifications (10×, 40×). It was found thatpyrene/Methocel nanosuspensions did not achieve uniform distribution inCVM and strongly aggregated into domains much larger than the mucus meshsize (data not shown). Such aggregation is indicative of mucoadhesivebehavior and effectively prevents mucus penetration. Therefore, furtherquantitative analysis of particle mobility was deemed unnecessary.Similarly to the positive control, all other tested pyrene/stabilizersystems achieved a fairly uniform distribution in CVM. Multiple particletracking confirmed that in all tested samples the negative controls werehighly constrained, while the positive controls were highly mobile asdemonstrated by <V_(mean)> for the positive controls being significantlygreater than those for the negative controls (Table 4).

TABLE 4 Ensemble-average velocity <V_(mean)> (um/s) and relative samplevelocity <V_(mean)>_(rel) for pyrene/stabilizer nanoparticles (sample)and controls in CVM. Negative Control Positive Control Sample Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD Pluronic F127 0.58 0.18 5.97 0.54 6.25 0.72 1.050.18 Pluronic F108 0.43 0.64 5.04 1.88 4.99 1.47 0.99 0.55 Pluronic P1050.56 0.52 4.38 1.36 4.47 2.11 1.02 0.69 Pluronic P103 0.58 0.77 4.5 2.014.24 1.95 0.93 0.74 Pluronic P123 0.56 0.44 4.56 1.44 3.99 1.66 0.860.54 Pluronic L121 0.42 0.3 4.27 2.04 0.81 0.51 0.10 0.16 Pluronic F680.56 0.52 4.38 1.36 0.81 0.7 0.07 0.23 Pluronic P65 0.26 0.25 4.52 2.150.53 0.56 0.06 0.15 Pluronic F87 0.95 1.6 5.06 1.34 0.74 0.78 −0.05−0.43 Pluronic F38 0.26 0.1 5.73 0.84 0.54 0.29 0.05 0.06 Kollicoat IR0.62 0.62 5.39 0.55 0.92 0.81 0.06 0.22 Kollidon 17 1.69 1.8 5.43 0.980.82 0.59 −0.23 −0.52 Kollidon 25 0.41 0.34 5.04 0.64 1.29 1.09 0.190.25 Kollindon 30 0.4 0.2 4.28 0.57 0.35 0.11 −0.01 0.06 Methocel E50**Methocel K100** Tween 20 0.77 0.93 5.35 1.76 1.58 2.02 0.18 0.49 Tween80 0.46 0.34 3.35 1.89 0.94 0.5 0.17 0.24 Solutol HS 0.42 0.13 3.49 0.50.8 0.6 0.12 0.20 Triton X100 0.26 0.13 4.06 1.11 0.61 0.19 0.09 0.07Tyloxapol 0.5 0.5 3.94 0.58 0.42 0.23 −0.02 −0.16 Cremophor RH40 0.480.21 3.2 0.97 0.49 0.24 0.00 0.12 SDS 0.3 0.12 5.99 0.84 0.34 0.15 0.010.03 CTAB 0.39 0.09 4.75 1.79 0.32 0.31 −0.02 −0.07 *Did not producestable nanosuspensions, hence not mucus-penetrating (velocity in CVM notmeasured) **Aggregated in CVM, hence not mucus-penetrating (velocity inCVM not measured)

It was discovered that nanoparticles obtained in the presence of certain(but, importantly, not all) surface-altering agents migrate through CVMat the same rate or nearly the same velocity as the positive control.Specifically, pyrene nanoparticles stabilized with Pluronics® F127,F108, P123, P105, and P103 exhibited <V_(mean)> that exceeded those ofthe negative controls by approximately an order of magnitude and wereindistinguishable, within experimental error, from those of the positivecontrols, as shown in Table 4 and FIG. 2A. For these samples,<V_(mean)>_(rel) values exceeded 0.5, as shown in FIG. 2B.

On the other hand, pyrene nanoparticles obtained with the othersurface-altering agents were predominantly or completely immobilized asdemonstrated by respective <V_(mean)>_(rel) values of no greater than0.4 and, with most surface-altering agents, no greater than 0.1 (Table 4and FIG. 2B). Additionally, FIGS. 3A-3D are histograms showingdistribution of V_(mean) within an ensemble of particles. Thesehistograms illustrate muco-diffusive behavior of samples stabilized withPluronic® F127 and Pluronic® F108 (similar histograms were obtained forsamples stabilized with Pluronic® P123, P105, and P103, but are notshown here) as opposed to muco-adhesive behavior of samples stabilizedwith Pluronic® 87, and Kollidon 25 (chosen as representativemuco-adhesive samples).

To identify the characteristics of Pluronics® that render pyrenenanocrystals mucus penetrating, <V_(mean)>_(rel) of the Pyrene/Pluronic®nanocrystals was mapped with respect to molecular weight of the PPOblock and the PEO weight content (%) of the Pluronics® used (FIG. 4). Itwas concluded that at least those Pluronics® that have the PPO block ofat least 3 kDa and the PEO content of at least about 30 wt % renderedthe nanocrystals mucus-penetrating. Without wishing to be bound by anytheory, it is believed that the hydrophobic PPO block can provideeffective association with the surface of the core particles if themolecular weight of that block is sufficient (e.g., at least about 3 kDain some embodiments); while the hydrophilic PEO blocks are present atthe surface of the coated particles and can shield the coated particlesfrom adhesive interactions with mucin fibers if the PEO content of thePluronic® is sufficient (e.g., at least 30 wt % in some embodiments). Asdescribed herein, in some embodiments the PEO content of thesurface-altering agent may be chosen to be greater than or equal toabout 10 wt % (e.g., at least about 15 wt %, or at least about 20 wt %),as a 10 wt % PEO portion rendered the particles mucoadhesive.

Example 2

This example describes the formation of mucus-penetrating particlesusing various non-polymeric solid particles.

The technique described in Example 1 was applied to other non-polymericsolid particles to show the versatility of the approach. F127 was usedas the surface-altering agent for coating a variety of activepharmaceuticals used as core particles. Sodium dodecyl sulfate (SDS) waschosen as a negative control so that each drug was compared to asimilarly sized nanoparticle of the same compound. An aqueous dispersioncontaining the pharmaceutical agent and Pluronic® F127 or SDS was milledwith milling media until particle size was reduced below 300 nm. Table 5lists the particle sizes for a representative selection of drugs thatwere milled using this method.

TABLE 5 Particle sizes for a representative selection of drugs milled inthe presence of SDS and F127. Drug Stabilizer Z-Ave D (nm) PDIFluticasone F127 203 0.114 propionate SDS 202 0.193 Furosemide F127 2170.119 SDS 200 0.146 Itraconazole F127 155 0.158 SDS 168 0.163Prednisolone F127 273 0.090 SDS 245 0.120 Loteprednol etabonate F127 2410.123 SDS 241 0.130 Budesonide F127 173 0.112 SDS 194 0.135 IndomethacinF127 225 0.123 SDS 216 0.154

In order to measure the ability of drug nanoparticles to penetrate mucusa new assay was developed which measures the mass transport ofnanoparticles into a mucus sample. Most drugs are not naturallyfluorescent and are therefore difficult to measure with particletracking microscopy techniques. The newly-developed bulk transport assaydoes not require the analyzed particles to be fluorescent or labeledwith dye. In this method, 20 μL of CVM is collected in a capillary tubeand one end is sealed with clay. The open end of the capillary tube isthen submerged in 20 μL of an aqueous suspension of particles which is0.5% w/v drug. After the desired time, typically 18 hours, the capillarytube is removed from the suspension and the outside is wiped clean. Thecapillary containing the mucus sample is placed in an ultracentrifugetube. Extraction media is added to the tube and incubated for 1 hourwhile mixing which removes the mucus from the capillary tube andextracts the drug from the mucus. The sample is then spun to removemucins and other non-soluble components. The amount of drug in theextracted sample can then be quantified using HPLC. The results of theseexperiments are in good agreement with those of the microscopy method,showing clear differentiation in transport between mucus penetratingparticles and conventional particles. The transport results for arepresentative selection of drugs are shown in FIG. 5. These resultscorroborate microscopy/particle tracking findings with Pyrene anddemonstrate the extension to common active pharmaceutical compounds;coating non-polymeric solid nanoparticles with F127 enhances mucuspenetration.

In Examples 1-2, 4-6, and 10, cervicovaginal mucus (CVM) samples wereobtained from healthy females volunteers age 18 years or older. CVM wascollected by inserting a Softcup® menstrual collection cup into thevaginal tract as described by the product literature for between 30seconds and 2 minutes. After removal the CVM was then collected from theSoftcup® by gentle centrifugation at ˜30×G to ˜120×G in a 50 mLcentrifuge tube. In Example 1, CVM was used undiluted and fresh (storedfor no longer than 7 days under refrigerated conditions). Barrier andtransport of all CVM samples used in Example 1 were verified withnegative (200 nm carboxylated polystyrene particles) and positive (200nm polystyrene particles modified with PEG 5K) controls. In Example 2,CVM was lyophilized and reconstituted. In Example 2, mucus was frozen at−50° C. and then lyophilized to dryness. Samples were then stored at−50° C. Before use, the mucus was reconstituted by grinding the solidinto a fine powder using a mortar and pestle followed by water additionto a final volume equal to the original volume to 2 times the originalvolume. The reconstituted mucus was then incubated at 4° C. for 12 hoursand used as described in Example 2. Barrier and transport of all CVMsamples used in Example 2 were verified with negative (200 nmcarboxylated polystyrene particles) and positive (F127 coated 200 nmpolystyrene particles) controls.

Example 3

This example describes the formation of mucus-penetrating particlesusing a core comprising the drug loteprednol etabonate (LE).

In order to demonstrate the value of enhanced mucus penetration in thedelivery of non-polymeric solid particles, an MPP formulation ofloteprednol etabonate (LE MPP; LE particles coated with Pluronic® F127made by the method described in Example 2) was compared to the currentlymarketed formulation, Lotemax®. Lotemax® is a steroid eye drop approvedfor the treatment of surface ocular inflammation. Conventionalparticles, such as those in Lotemax®, are extensively trapped by theperipheral rapidly-cleared mucus layer in the eye and, hence, are alsorapidly cleared. LE MPPs are able to avoid adhesion to, and effectivelypenetrate through, mucus to facilitate sustained drug release directlyto underlying tissues. Enhancing drug exposure at the target site wouldallow the overall dose to be reduced, increasing patient compliance andsafety. In vivo, a single topical instillation of LE MPP to New Zealandwhite rabbits produced significantly higher drug levels in palpebralconjunctiva, bulbar conjunctiva, and cornea compared to an equivalentdose of Lotemax® (FIGS. 6A-6C). At 2 hours LE levels from MPP are 6, 3,and 8 times higher than from Lotemax® (palpebral, bulbar, and cornea,respectively). Notably, LE levels from MPP are approximately 2 timeshigher at 2 hours than levels from Lotemax® at 30 minutes. These resultsdemonstrate the enhanced exposure achievable with the MPP technologyover the commercial formulation.

Example 4

The following describes a non-limiting example of a method of formingmucus-penetrating particles from pre-fabricated polymeric particles byphysical adsorption of certain poly(vinyl alcohol) polymers (PVA).Carboxylated polystyrene nanoparticles (PSCOO) were used as theprefabricated particle/core particle with a well-established stronglymucoadhesive behavior. The PVAs acted as surface-altering agents formingcoatings around the core particles. PVA of various molecular weights(MW) and hydrolysis degrees were evaluated to determine effectiveness ofthe coated particles in penetrating mucus.

PSCOO particles were incubated in aqueous solution in the presence ofvarious PVA polymers to determine whether certain PVAs can physically(non-covalently) coat the core particle with a mucoinert coating thatwould minimize particle interactions with mucus constituents and lead torapid particle penetration in mucus. In these experiments, the PVA actedas a coating around the core particles, and the resulting particles weretested for their mobility in mucus, although in other embodiments, PVAmay be exchanged with other surface-altering agents that can increasemobility of the particles in mucus. The PVAs tested ranged in theaverage molecular weight from 2 kDa to 130 kDa and in the averagehydrolysis degree from 75% to 99+%. The PVAs that were tested are listedin Table 1, shown above.

The particle modification process was as follows: 200 nmcarboxylated-modified red fluorescent polystyrene nanoparticles (PSCOO)were purchased from Invitrogen. The PSCOO particles (0.4-0.5 wt %) wereincubated in an aqueous PVA solution (0.4-0.5 wt %) for at least 1 hourat room temperature.

The mobility and distribution of the modified nanoparticles in humancervicovaginal mucus (CVM) were characterized using fluorescencemicroscopy and multiple particle tracking software. In a typicalexperiment, <1.5 μL of an incubated nanosuspension (diluted ˜10× with0.5 wt % aqueous solution of a corresponding PVA) was added to 20 μl offresh CVM along with controls. Conventional nanoparticles (200 nm bluefluorescent carboxylate-modified polystyrene microspheres fromInvitrogen) were used as a negative control to confirm the barrierproperties of the CVM samples. Yellow-green fluorescent polystyrenenanoparticles covalently coated with PEG 2 kDa were used as a positivecontrol with well-established MPP behavior. Using a fluorescentmicroscope equipped with a CCD camera, 15 s movies were captured at atemporal resolution of 66.7 ms (15 frames/s) under 100× magnificationfrom several areas within each sample for each type of particles: sample(observed through a Texas Red filter set), negative control (observedthrough a DAPI filter set), and positive control (observed through aFITC filter set). Next, using an advanced image processing software,individual trajectories of multiple particles were measured over atime-scale of at least 3.335 s (50 frames). Resulting transport data arepresented here in the form of trajectory-mean velocity V_(mean), i.e.,velocity of an individual particle averaged over its trajectory, andensemble-average velocity <V_(mean)>, i.e., V_(mean) averaged over anensemble of particles. To enable easy comparison between differentsamples and normalize velocity data with respect to natural variabilityin penetrability of CVM samples, ensemble-average (absolute) velocity isthen converted to relative sample velocity <V_(mean)>_(rel) according tothe formula shown in Equation 1. Multiple particle tracking confirmedthat in all tested CVM samples the negative controls were constrained,while the positive controls were mobile as demonstrated by thedifferences in <V_(mean)> for the positive and negative controls (Table6).

TABLE 6 Transport of nanoparticles incubated with various PVA (sample)and controls in CVM: Ensemble-average velocity <V_(mean)> (μm/s) andrelative sample velocity <V_(mean)>_(rel). Sample Negative ControlPositive Control Sample (relative) Stabilizer <V_(mean)> SD <V_(mean)>SD <V_(mean)> SD <V_(mean)>_(rel) SD PVA2K75 1.39 0.33 3.3 0.68 3.44 0.71.07 0.59 PVA9K80 0.4 0.08 5.13 1.16 4.88 1.74 0.95 0.44 PVA13K87 0.560.61 5.23 1.24 4.92 1.77 0.93 0.49 PVA31K87 0.53 0.63 4.48 1.38 3.691.94 0.80 0.60 PVA57K86 0.5 0.25 5.74 1.11 4.76 0.91 0.81 0.25 PVA85K870.29 0.28 4.25 0.97 4.01 0.71 0.94 0.31 PVA105K80 0.98 0.52 5.44 0.864.93 0.66 0.89 0.27 PVA130K87 1.41 0.56 3.75 0.82 3.57 0.6 0.92 0.53PVA95K95 0.51 0.36 3.19 0.68 0.45 0.19 −0.02 −0.15 PVA13K98 0.43 0.173.42 1.65 0.5 0.76 0.02 0.26 PVA31K98 0.41 0.23 6.03 1.19 0.26 0.14−0.03 −0.05 PVA85K99 0.28 0.1 4.7 0.82 0.53 0.77 0.06 0.18

It was discovered that nanoparticles incubated in the presence ofcertain (but, interestingly, not all) PVA transported through CVM at thesame rate or nearly the same velocity as the positive control.Specifically, the particles stabilized with PVA2K75, PVA9K80, PVA13K87,PVA31K87, PVA57K86, PVA85K87, PVA105K80, and PVA130K87 exhibited<V_(mean)> that significantly exceeded those of the negative controlsand were indistinguishable, within experimental error, from those of thepositive controls. The results are shown in Table 6 and FIG. 7A. Forthese samples, <V_(mean)>_(rel) values exceeded 0.5, as shown in FIG.7B.

On the other hand, nanoparticles incubated with PVA95K95, PVA13K98,PVA31K98, and PVA85K99 were predominantly or completely immobilized asdemonstrated by respective <V_(mean)>_(rel) values of no greater than0.1 (Table 6 and FIG. 7B).

To identify the characteristics of the PVA that render particles mucuspenetrating, <V_(mean)>_(rel) of the nanoparticles prepared byincubation with the various PVAs was mapped with respect to MW andhydrolysis degree of the PVAs used (FIG. 8). It was concluded that atleast those PVAs that have the hydrolysis degree of less than 95%rendered the nanocrystals mucus-penetrating. Without wishing to be boundby any theory, it is believed that the unhydrolyzed (vinyl acetate)units of PVA can provide effective hydrophobic association with thesurface of the core particles if the content of these segments in thePVA is sufficient (e.g., greater than 5% in some embodiments); while thehydrophilic (vinyl alcohol) units of PVA present at the surface of thecoated particles render them hydrophilic and can shield the coatedparticles from adhesive interactions with mucus.

To further confirm the ability of the specific PVA grades to convertmucoadhesive particles into mucus-penetrating particles by physicaladsorption, PSCOO nanoparticles incubated with the various PVAs weretested using the bulk transport assay. In this method, 20 μL of CVM wascollected in a capillary tube and one end is sealed with clay. The openend of the capillary tube is then submerged in 20 μL of an aqueoussuspension of particles which is 0.5% w/v drug. After the desired time,typically 18 hours, the capillary tube is removed from the suspensionand the outside is wiped clean. The capillary containing the mucussample is placed in an ultracentrifuge tube. Extraction media is addedto the tube and incubated for 1 hour while mixing which removes themucus from the capillary tube and extracts the drug from the mucus. Thesample is then spun to remove mucins and other non-soluble components.The amount of drug in the extracted sample can then be quantified usingHPLC. The results of these experiments are in good agreement with thoseof the microscopy method, showing clear differentiation in transportbetween positive (mucus-penetrating particles) and negative controls(conventional particles). The bulk transport results for PSCOOnanoparticles incubated with the various PVAs are shown in FIG. 9. Theseresults corroborate microscopy/particle tracking findings with PSCOOnanoparticles incubated with the various PVAs and demonstrate theincubating nanoparticles with partially hydrolyzed PVAs enhances mucuspenetration.

Example 5

The following describes a non-limiting example of a method of formingmucus-penetrating particles by an emulsification process in the presenceof certain poly(vinyl alcohol) polymers (PVA). Polylactide (PLA), abiodegradable pharmaceutically relevant polymer was used as a materialto form the core particle via an oil-in-water emulsification process.The PVAs acted as emulsion surface-altering agents and surface-alteringagents forming coatings around the produced core particles. PVA ofvarious molecular weights (MW) and hydrolysis degrees were evaluated todetermine effectiveness of the formed particles in penetrating mucus.

PLA solution in dichloromethane was emulsified in aqueous solution inthe presence of various PVA to determine whether certain PVAs canphysically (non-covalently) coat the surface of generated nanoparticleswith a coating that would lead to rapid particle penetration in mucus.In these experiments, the PVA acted as an surfactant that forms astabilizing coating around droplets of emulsified organic phase that,upon solidification, form the core particles The resulting particleswere tested for their mobility in mucus, although in other embodiments,PVA may be exchanged with other surface-altering agents that canincrease mobility of the particles in mucus. The PVAs tested ranged inthe average molecular weight from 2 kDa to 130 kDa and in the averagehydrolysis degree from 75% to 99+%. The PVAs that were tested are listedin Table 1, shown above.

The emulsification-solvent evaporation process was as follows:Approximately 0.5 mL of 20-40 mg/ml solution of PLA (Polylactide grade100DL7A, purchased from Surmodics) in dichloromethane was emulsified inapproximately 4 mL of an aqueous PVA solution (0.5-2 wt %) by sonicationto obtain a stable emulsion with the target number-average particle sizeof <500 nm. Obtained emulsions were immediately subjected to exhaustiverotary evaporation under reduced pressure at room temperature to removethe organic solvent. Obtained suspensions were filtered through 1 micronglass fiber filters to remove any agglomerates. Table 7 lists theparticle size characteristics of the nanosuspensions obtained by thisemulsification procedure with the various PVA. In all cases, afluorescent organic dye Nile Red was added to the emulsified organicphase to fluorescently label the resulting particles.

TABLE 7 Particle size measured by DLS in nanosuspensions obtained by theemulsification process with various PVA. PVA Grade Z-Ave D (nm) N-Ave D(nm) PVA2K75 186 156 PVA10K80 208 173 PVA13K98 245 205 PVA31K87 266 214PVA31K98 245 228 PVA85K87 356 301 PVA85K99 446 277 PVA95K95 354 301PVA105K80 361 300 PVA130K87 293 243

The mobility and distribution of the produced nanoparticles in humancervicovaginal mucus (CVM) were characterized using fluorescencemicroscopy and multiple particle tracking software. In a typicalexperiment, ≤1.5 uL of a nanosuspension (diluted if necessary to the PVAconcentration of ˜0.5%) was added to 20 μl of fresh CVM along withcontrols. Conventional nanoparticles (200 nm blue fluorescentcarboxylate-modified polystyrene microspheres from Invitrogen) were usedas a negative control to confirm the barrier properties of the CVMsamples. Yellow-green fluorescent polystyrene nanoparticles covalentlycoated with PEG 2 kDa were used as a positive control withwell-established MPP behavior. Using a fluorescent microscope equippedwith a CCD camera, 15 s movies were captured at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample (observed through a TexasRed filter set due to the encapsulated Nile Red), negative control(observed through a DAPI filter set), and positive control (observedthrough a FITC filter set). Next, using an advanced image processingsoftware, individual trajectories of multiple particles were measuredover a time-scale of at least 3.335 s (50 frames). Resulting transportdata are presented here in the form of trajectory-mean velocityV_(mean), i.e., velocity of an individual particle averaged over itstrajectory, and ensemble-average velocity <V_(mean)>, V_(mean) averagedover an ensemble of particles. To enable easy comparison betweendifferent samples and normalize velocity data with respect to naturalvariability in penetrability of CVM samples, ensemble-average (absolute)velocity is then converted to relative sample velocity <V_(mean)>_(rel)according to the formula shown in Equation 1. Multiple particle trackingconfirmed that in all tested CVM samples the negative controls wereconstrained, while the positive controls were mobile as demonstrated bythe differences in <V_(mean)> for the positive and negative controls(Table 8).

TABLE 8 Transport of PLA nanoparticles obtained by the emulsificationprocess with various PVAs (sample) and controls in CVM: Ensemble-averagevelocity <V_(mean)> (um/s) and relative sample velocity<V_(mean)>_(rel). Sample Negative Control Positive Control Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD PVA2K75 0.95 0.64 5.5 0.92 5.51 1.2 1.00 0.39PVA9K80 0.72 0.47 5.61 0.79 4.6 1.5 0.79 0.35 PVA31K87 0.63 0.60 4.941.50 3.36 1.84 0.63 0.51 PVA85K87 0.57 0.4 4.49 1.21 2.9 1.56 0.59 0.45PVA105K80 0.69 0.56 4.85 1.54 3.55 1.26 0.69 0.43 PVA130K87 0.95 0.544.98 1.25 3.46 1.23 0.62 0.39 PVA95K95 1.39 1.28 5.72 1.57 1.63 1.5 0.060.46 PVA13K98 1.02 0.49 5.09 0.99 2.61 1.54 0.39 0.41 PVA31K98 1.09 0.65.09 0.9 2.6 1.13 0.38 0.34 PVA85K99 0.47 0.33 5.04 2.2 0.81 0.77 0.070.19

It was discovered that nanoparticles prepared in the presence of certain(but, interestingly, not all) PVA transported through CVM at the samerate or nearly the same velocity as the positive control. Specifically,the particles stabilized with PVA2K75, PVA9K80, PVA13K87, PVA31K87,PVA85K87, PVA105K80, and PVA130K87 exhibited <V_(mean)> thatsignificantly exceeded those of the negative controls and wereindistinguishable, within experimental error, from those of the positivecontrols, as shown in Table 8 and FIG. 10A. For these samples,<V_(mean)>_(rel) values exceeded 0.5, as shown in FIG. 10B.

On the other hand, pyrene nanoparticles obtained with PVA95K95,PVA13K98, PVA31K98, and PVA85K99 were predominantly or completelyimmobilized as demonstrated by respective <V_(mean)>_(rel) values of nogreater than 0.4 (Table 8 and FIG. 10B). To identify the characteristicsof the PVA that render particles mucus penetrating, <V_(mean)>_(rel) ofthe nanoparticles prepared with the various PVAs was mapped with respectto MW and hydrolysis degree of the PVAs used (Table 6 and FIG. 7B). Itwas concluded that at least those PVAs that have the hydrolysis degreeof less than 95% rendered the nanocrystals mucus-penetrating. Withoutwishing to be bound by any theory, it is believed that the unhydrolyzed(vinyl acetate) units of PVA can provide effective hydrophobicassociation with the surface of the core particles if the content ofthese segments in the PVA is sufficient (e.g., greater than 5% in someembodiments); while the hydrophilic (vinyl alcohol) units of PVA presentat the surface of the coated particles render them hydrophilic and canshield the coated particles from adhesive interactions with mucus.

Example 6

The following describes a non-limiting example of a method of formingmucus-penetrating non-polymeric solid particles by milling in thepresence of certain poly(vinyl alcohol) polymers (PVA). Pyrene, a modelhydrophobic compound, was used as the core particle processed by amilling. The PVA acted as milling aids facilitating particle sizereduction of the core particles and surface-altering agents formingcoatings around the core particles. PVA of various molecular weights(MW) and hydrolysis degrees were evaluated to determine effectiveness ofthe milled particles in penetrating mucus.

Pyrene was milled in aqueous dispersions in the presence of various PVAto determine whether PVAs of certain MW and hydrolysis degree can: 1)aid particle size reduction to several hundreds of nanometers and 2)physically (non-covalently) coat the surface of generated nanoparticleswith a mucoinert coating that would minimize particle interactions withmucus constituents and prevent mucus adhesion. In these experiments, thePVA acted as a coating around the core particles, and the resultingparticles were tested for their mobility in mucus. The PVAs testedranged in the average molecular weight from 2 kDa to 130 kDa and in theaverage hydrolysis degree from 75% to 99+%. The PVAs that were testedare listed in Table 1, shown above. A variety of other polymers,oligomers, and small molecules listed in Table, includingpharmaceutically relevant excipients such as polyvinylpyrrolidones(Kollidon), hydroxypropyl methylcellulose (Methocel), Tween, Span, etc.,were tested in a similar manner.

TABLE 9 Other surface-altering agents tested with pyrene as a modelcompound. Chemical Family Grades Polyvinylpyrrolidone (PVP) Kollidon 17Kollidon 25 Kollindon 30 PVA-poly(ethylene glycol) Kollicoat IRgraft-copolymer Hydroxypropyl Methocel E50 methylcellulose (HPMC)Methocel K100 Non-ionic polyoxyethylene Solutol HS 15 surfactants Span20 Span 80 Triton X100 Tween 20 Tween 80 Tyloxapol Non-ionic small Octylglucoside molecule surfactants Ionic small Cetytrimethylammoniummolecule surfactants bromide (CTAB) Sodium dodecyl sulfate (SDS)

An aqueous dispersion containing pyrene and one of the surface-alteringagents listed above was stirred with milling media until particle sizewas reduced below 500 nm (as measured by dynamic light scattering).Table 10 lists particle size characteristics of pyrene particlesobtained by milling in the presence of the various surface-alteringagents. When Span 20, Span 80, or Octyl glucoside was used assurface-altering agents, stable nanosuspensions could not be obtained.Therefore, these surface-altering agents were excluded from furtherinvestigation due to their inability to effectively aid particle sizereduction.

TABLE 10 Particle size measured by DLS in nanosuspensions obtained bymilling of pyrene with various surface-altering agents. Stabilizer Z-AveD (nm) N-Ave D (nm) PVA2K75 340 301 PVA9K80 380 337 PVA13K87 375 326PVA13K98 396 314 PVA31K87 430 373 PVA31K98 344 220 PVA85K87 543 434PVA85K99 381 236 PVA95K95 534 392 PVA130K87 496 450 Kollidon 17 237 163Kollidon 25 307 210 Kollindon 30 255 185 Kollicoat IR 364 192 MethocelE50 244 160 Methocel K100 375 216 Tween 20 567 381 Tween 80 553 322Solutol HS 576 378 Triton X100 410 305 Tyloxapol 334 234 Cremophor RH40404 373 Span 20 not measurable* Span 80 not measurable* Octyl glucosidenot measurable* SDS 603 377 CTAB 432 354 *milling with Span 20, Span 80,Octyl glucoside failed to effectively reduce pyrene particle size andproduce stable nanosuspensions.

The mobility and distribution of the produced pyrene nanoparticles inhuman cervicovaginal mucus (CVM) were characterized using fluorescencemicroscopy and multiple particle tracking software. In a typicalexperiment, ≤1.5 uL of a nanosuspension (diluted if necessary to thesurfactant concentration of ˜1%) was added to 20 μl of fresh CVM alongwith controls. Conventional nanoparticles (200 nm yellow-greenfluorescent carboxylate-modified polystyrene microspheres fromInvitrogen) were used as a negative control to confirm the barrierproperties of the CVM samples. Red fluorescent polystyrene nanoparticlescovalently coated with PEG 5 kDa were used as a positive control withwell-established MPP behavior. Using a fluorescent microscope equippedwith a CCD camera, 15 s movies were captured at a temporal resolution of66.7 ms (15 frames/s) under 100× magnification from several areas withineach sample for each type of particles: sample (pyrene), negativecontrol, and positive control (natural blue fluorescence of pyreneallowed observing of pyrene nanoparticles separately from the controls).Next, using an advanced image processing software, individualtrajectories of multiple particles were measured over a time-scale of atleast 3.335 s (50 frames). Resulting transport data are presented herein the form of trajectory-mean velocity V_(mean), i.e., velocity of anindividual particle averaged over its trajectory, and ensemble-averagevelocity <V_(mean)>, i.e., V_(mean) averaged over an ensemble ofparticles. To enable easy comparison between different samples andnormalize velocity data with respect to natural variability inpenetrability of CVM samples, ensemble-average (absolute) velocity isthen converted to relative sample velocity <V_(mean)>_(rel) according tothe formula shown in Equation 1.

Prior to quantifying mobility of pyrene particles, their spatialdistribution in the mucus sample was assessed visually. It was foundthat pyrene/Methocel nanosuspensions did not achieve uniformdistribution in CVM and strongly aggregated into domains much largerthan the mucus mesh size (data not shown). Such aggregation isindicative of mucoadhesive behavior and effectively prevents mucuspenetration. Therefore, further quantitative analysis of particlemobility was deemed unnecessary. Similarly to the positive control, allother tested pyrene/stabilizer systems achieved a fairly uniformdistribution in CVM. Multiple particle tracking confirmed that in alltested CVM samples the negative controls were constrained, while thepositive controls were mobile as demonstrated by the differences in<V_(mean)> for the positive and negative controls (Table).

TABLE 11 Transport of pyrene nanoparticles (sample) obtained withvarious surface-altering agents and controls in CVM: Ensemble-averagevelocity <V_(mean)> (um/s) and relative sample velocity<V_(mean)>_(rel). Sample Negative Control Positive Control Sample(relative) Stabilizer <V_(mean)> SD <V_(mean)> SD <V_(mean)> SD<V_(mean)>_(rel) SD PVA2K75 0.4 0.24 5.73 0.73 4.73 1.08 0.81 0.24PVA9K80 0.36 0.20 6.00 0.70 6.19 1.13 1.03 0.24 PVA13K87 1.01 1.21 5.090.98 4.54 1.03 0.87 0.51 PVA31K87 1.28 1.14 4.88 0.6 4.57 1.123 0.910.55 PVA85K87 1.05 0.9 4.1 0.57 3.3 0.98 0.74 0.51 PVA130K87 0.51 0.825.29 0.73 4.12 1.49 0.76 0.40 PVA95K95 0.4 0.27 4.53 1.03 0.67 0.6 0.070.16 PVA13K98 0.61 0.42 2.13 0.99 1.29 0.57 0.45 0.56 PVA31K98 0.68 0.875.77 1.24 2.69 2.02 0.39 0.45 PVA85K99 0.43 0.23 5.42 0.97 2.23 1.600.36 0.33 Kollicoat IR 0.62 0.62 5.39 0.55 0.92 0.81 0.06 0.22 Kollidon17 1.69 1.8 5.43 0.98 0.82 0.59 −0.23 −0.52 Kollidon 25 0.41 0.34 5.040.64 1.29 1.09 0.19 0.25 Kollindon 30 0.4 0.2 4.28 0.57 0.35 0.11 −0.010.06 Methocel E50* Methocel K100* Tween 20 0.77 0.93 5.35 1.76 1.58 2.020.18 0.49 Tween 80 0.46 0.34 3.35 1.89 0.94 0.5 0.17 0.24 Solutol HS0.42 0.13 3.49 0.5 0.8 0.6 0.12 0.20 Triton X100 0.26 0.13 4.06 1.110.61 0.19 0.09 0.07 Tyloxapol 0.5 0.5 3.94 0.58 0.42 0.23 −0.02 −0.16Cremophor RH40 0.48 0.21 3.2 0.97 0.49 0.24 0.00 0.12 SDS 0.3 0.12 5.990.84 0.34 0.15 0.01 0.03 CTAB 0.39 0.09 4.75 1.79 0.32 0.31 −0.02 −0.07*Aggregated in CVM, hence not mucus-penetrating (velocity in CVM notmeasured)

It was discovered that nanoparticles obtained in the presence of certain(but, interestingly, not all) PVA transported through CVM at the samerate or nearly the same velocity as the positive control. Specifically,pyrene nanoparticles stabilized with PVA2K75, PVA9K80, PVA13K87,PVA31K87, PVA85K87, and PVA130K87 exhibited <V_(mean)> thatsignificantly exceeded those of the negative controls and wereindistinguishable, within experimental error, from those of the positivecontrols, as shown in Table 11 and FIG. 12A. For these samples,<V_(mean)>_(rel) values exceeded 0.5, as shown in FIG. 12B.

On the other hand, pyrene nanoparticles obtained with the othersurface-altering agents, including PVA95K95, PVA13K98, PVA31K98, andPVA85K99, were predominantly or completely immobilized as demonstratedby respective <V_(mean)>_(rel) values of no greater than 0.5 and, withmost surface-altering agents, no greater than 0.4 (Table 11 and FIG. 12B). Additionally, FIGS. 13A-13F are histograms showing distribution ofV_(mean) within an ensemble of particles. These histograms illustratemuco-diffusive behavior of samples stabilized with PVA2K75 and PVA9K80(similar histograms were obtained for samples stabilized with PVA13K87,PVA31K87, PVA85K87, and PVA130K87, but are not shown here) as opposed tomuco-adhesive behavior of samples stabilized with PVA31K98, PVA85K99,Kollidon 25, and Kollicoat IR (chosen as representative muco-adhesivesamples).

To identify the characteristics of the PVA that render pyrenenanocrystals mucus penetrating, <V_(mean)>_(rel) of the pyrenenanocrystals stabilized with various PVAs was mapped with respect to MWand hydrolysis degree of the PVAs used (FIG. 14). It was concluded thatat least those PVAs that have the hydrolysis degree of less than 95%rendered the nanocrystals mucus-penetrating. Without wishing to be boundby any theory, it is believed that the unhydrolyzed (vinyl acetate)segments of PVA can provide effective hydrophobic association with thesurface of the core particles if the content of these segments in thePVA is sufficient (e.g., greater than 5% in some embodiments); while thehydrophilic (vinyl alcohol) segments of PVA present at the surface ofthe coated particles render them hydrophilic and can shield the coatedparticles from adhesive interactions with mucus.

Example 7

This example shows an improved ophthalmic delivery of a pharmaceuticalagent from mucus-penetrating particles comprised of a polymeric coreencapsulating the pharmaceutical agent and mucus-penetrating particlescomprised of a drug core without any polymeric carrier.

In order to demonstrate the value of enhanced mucus penetration in thedelivery of pharmaceutical agents to the eye, concentrations ofloteprednol etabonate in the cornea of New Zealand White Rabbits weremeasured following a single equivalent dose of Lotemax® (the currentlymarketed ophthalmic suspension of loteprednol etabonate (LE), a softsteroid indicated for the treatment of ocular inflammation), MPP1(mucus-penetrating particles comprised of a polymeric core encapsulatingLE), and MPP2 (mucus-penetrating particles comprised of the LE core).MPP1 particles were made by nanoprecipitation of loteprednol etabonatewith poly(lactide) (100DL2A from Surmodics) from a solution in acetoneinto an aqueous solution. MPP2 particles were made by the methoddescribed in Example 2. Both MPP1 and MPP2 particles were coated withPluronic® F127. As shown in FIG. 17A and FIG. 17B, MPP1 and MPP2formulations resulted in higher drug levels in the cornea compared tothose from the commercial drop having the same concentration ofparticles as that of the MPP formulations. Without wishing to be boundby any theory, it is believed that conventional particles, such as thosein Lotemax®, are extensively trapped by the peripheral rapidly-clearedmucus layer in the eye and, hence, are rapidly cleared; while the MPPparticles are able to avoid adhesion to mucus and, hence, achieveprolonged residence at the ocular surface and facilitate sustained drugrelease directly to underlying tissues.

Example 8

This example shows an improved delivery of a pharmaceutical agent frommucus-penetrating particles coated with Pluronic® F127 to the back ofthe eye, including the retina, choroid and sclera, which is not seenwith conventional particles. Delivery to the cornea and iris was alsoimproved for particles coated with Pluronic® F127 compared conventionalparticles.

In order to demonstrate the value of enhanced mucus penetration in thedelivery of non-polymeric solid particles, an MPP formulation ofloteprednol etabonate (LE MPP; LE particles coated with Pluronic® F127made by the method described in Example 2) was compared to the currentlymarketed formulation, Lotemax®. Lotemax® is a steroid eye drop approvedfor the treatment of ocular inflammation. Conventional particles, suchas those in Lotemax®, are extensively trapped by the peripheralrapidly-cleared mucus layer in the eye and, hence, are rapidly cleared.LE MPP are able to avoid adhesion to, and effectively penetrate through,mucus to facilitate sustained drug release directly to underlyingtissues. Not only is delivery to the ocular surface enhanced asdescribed in Example 3, but delivery to the middle and back of the eyeis also enhanced. In vivo, a single topical instillation of LE MPP toNew Zealand white rabbits produced significantly higher drug levels incornea, iris/ciliary body, aqueous humor, retina, choroid, and scleracompared to an equivalent dose of Lotemax® (FIG. 18). Current commercialeye drops are used in the treatment of anterior ocular disorders, butare not effective in treating posterior disorders because drug does notreach the back of the eye. Here the retina, choroid and sclera aresampled using an 8 mm punch where the human macula would be located. Inthe back of the eye LE levels are below the limit of detection forLotemax®, while LE MPP deliver detectable levels of LE to retina,choroid, and sclera. These results demonstrate the utility of thenon-polymeric solid MPP approach over conventional approaches.

Example 9

This example describes the measurement of the density of Pluronic® F127on the surface of particles comprising a nanocrystal core of apharmaceutical agent.

An aqueous dispersion containing a pharmaceutical agent and Pluronic®F127 was milled with milling media until particle size was reduced below300 nm. A small volume from the milled suspension was diluted to anappropriate concentration (˜100 μg/mL, for example) and the z-averagediameter was taken as a representative measurement of particle size. Theremaining suspension was then divided into two aliquots. Using HPLC, thefirst aliquot was assayed for the total concentration of drug (here,loteprednol eltabonate or fluticasone propionate) and for the totalconcentration of surface-altering moiety (here, Pluronic® F127). Againusing HPLC the second aliquot was assayed for the concentration of freeor unbound surface-altering moiety. In order to get only the free orunbound surface-altering moiety from the second aliquot, the particles,and therefore any bound surface-altering moiety, were removed byultracentrifugation. By subtracting the concentration of the unboundsurface-altering moiety from the total concentration of surface-alteringmoiety, the concentration of bound surface-altering moiety wasdetermined. Since the total concentration of drug was also determinedfrom the first aliquot, the mass ratio between the core material and thesurface-altering moiety can be determined. Using the molecular weight ofthe surface-altering moiety, the number of surface-altering moiety tomass of core material can be calculated. To turn this number into asurface density measurement, the surface area per mass of core materialneeds to be calculated. The volume of the particle is approximated asthat of a sphere with the diameter obtained from DLS allowing for thecalculation of the surface area per mass of core material. In this waythe number of surface-altering moieties per surface area is determined.FIG. 19 shows the results of surface-moiety density determination forloteprednol Itabonate and fluticasone propionate.

Example 10

The following describes a non-limiting example of a method offormingmucus-penetrating particles using a core comprising the drugloteprednol etabonate (LE) by milling in the presence of variousPluronic® surface-altering agents.

LE was milled as an aqueous suspension with milling media and in thepresence of a stabilizer. Pluronics of various grades (listed in Table12) were tested as surface-altering agents in order to determine whethercertain Pluronic grades can: 1) aid reduction of particle size of LE tothe submicron range and 2) physically (non-covalently) coat the surfaceof generated LE nanoparticles with a mucoinert coating that wouldminimize particle interactions with mucus constituents and prevent mucusadhesion. In these experiments, the Pluronics acted as a coating aroundthe core particles, and the resulting particles were tested for theirmobility in mucus, although in other embodiments, the surface-alteringagents may be exchanged with other surface-altering agents that canincrease mobility of the particles in mucus.

The milling process was carried out until LE particles were small andpolydispersity was low (i.e., z-average particle diameter below 500 nmand polydispersity index <0.20 as measured by dynamic light scattering).Table 12 lists particle size characteristics of LE particles obtained bymilling in the presence of the various Pluronics®. Particle size wasmeasured by dynamic light scattering. Milling of LE in the presence ofPluronic® L31, L35, L44 or L81 failed to produce stable nanosuspensions.Therefore, these Pluronics were excluded from further investigation dueto their inability to effectively aid particle size reduction.

The mobility of LE nanoparticles from the produced nanosuspensions infresh undiluted human cervicovaginal mucus (CVM) was characterized bydark-field microscopy using a CytoViva® High Resolution IlluminationSystem which allows visualization of fluorescent and non-fluorescentnano-sized objects. In a typical experiment, 0.5 μL of a nanosuspensionwas added to 20 μL of undiluted CVM pre-deposited into a 20 μL well on amicroscope slide. Using a CCD camera, 15 s movies were captured at atemporal resolution of 66.7 ms (15 frames/s) under 100× magnificationfrom several randomly selected areas within each sample. Mobility ofparticles in the movies was scored on a scale from 0 to 3 in order ofincreasing mobility, in a single-blind experiment by independentobservers. The scoring criterion is as follows: 0-0.5 immobile; 0.51-1.5slightly mobile; 1.51-2.5 moderately mobile; and 2.51-3.0 very mobile.

The average mobility scores for each LE/Pluronic sample are plotted inFIG. 20 as a function of the molecular weight of the PPO component (MWPPO) and the weight percentage of the PEO component (% PEO) of thePluronic® polymer. It was discovered that LE milled in the presence ofPluronic® F87, F108, and F127 resulted in particles that are very mobilein CVM (mobility score >2.51). This corresponds to Pluronic® polymerswith physical properties of MW PPO≥2.3 kDa and % PEO≥70%. LEnanocrystals milled in Pluronic® P103, P105, and P123 are moderatelymobile (mobility score 1.51.-2.50). The corresponding physicalproperties of this Pluronic® class is MW PPO≥3.3 kDa and 30%≤% PEO≤70%.Pluronic® L121, P65, F38, and F68 produced LE nanocrystals that were notmobile (mobility score <0.50). This group of Pluronic® polymersconstitutes those with MW PPO≤1.9 kDa and % PEO≤10%. Pluronic® L31, L35,L44, or L81, whose physical properties also fall into theprevious-mentioned MW PPO and % PEO categories, failed to produce smalland monodisperse particles and are considered immobile (mobility score<0.50) in the analysis.

Without wishing to be bound by any theory, it is believed that thehydrophobic PPO block can provide effective association with the surfaceof the core LE particles if the molecular weight of that block issufficient (e.g., at least about 2.3 kDa in some embodiments); while thehydrophilic PEO blocks are present at the surface of the coated LEparticles and can shield the coated LE particles from adhesiveinteractions with mucin fibers if the PEO content of the Pluronic® issufficient (e.g., at least 30 wt % in some embodiments). As describedherein, in some embodiments the PEO content of the surface-alteringagent may be chosen to be greater than or equal to about 10 wt % (e.g.,at least about 15 wt % or at least about 20 wt %), as a 10 wt % PEOportion rendered the particles mucoadhesive.

Interestingly, the molecular weight of the PPO block needed to providehigh mobility (mobility score >2.51) when LE was used as the core was atleast about 2.3 kDa, compared to 3 kDa for when pyrene was used as thecore. This data suggests that the surface altering agent (e.g., themolecular weight of the PPO block) may be varied, depending on the coreto be coated, to tailor the mobility of the particle.

TABLE 12 Particle size measured by Dynamic Light Scattering (DLS) innanosuspensions obtained by milling of LE with various Pluronic ®surface-altering agents. Z-average diameter Pluronic ® grade (nm)Polydispersity index L31* not measurable* not measurable* L35* notmeasurable* not measurable* L44* not measurable* not measurable* L81*not measurable* not measurable* L101* not measurable* not measurable*L121 343 0.18 P65 224 0.06 P103 251 0.08 P105 356 0.19 P123 281 0.12 F38233 0.10 F68 404 0.13 F87 316 0.07 F108 294 0.12 F127 404 0.13 *denotessamples that failed to effectively reduce LE particle size and producestable nanosuspensions. For these samples, Z-Ave diameter was greaterthan at least 1 μm and not measurable with DLS.

Example 11

The following describes a non-limiting example of a method of formingmucus-penetrating particles (MPP) using a core comprising the drugloteprednol etabonate (LE) in the presence of other components, such asPluronic®, glycerin, sodium chloride (NaCl), disodiumethylenediaminetetraacetic acid (Na₂EDTA), and benzalkonium chloride(BAC).

The method of forming the loteprednol etabonate mucus-penetratingparticles (LE MPP) involved two consecutive steps: milling and dilution.In the milling step, a coarse aqueous suspension containing about 2-20%loteprednol etabonate (coarse or micronized crystals), about 0.2-20%Pluronic® F127, about 0.5-3% glycerin, about 0.1-1% sodium chloride, andabout 0.001-0.1% EDTA was milled in the presence of milling media toproduce a nanosuspension of loteprednol etabonate particles sized in therange of 200-300 nm.

In the subsequent dilution step, the obtained nanocrystalline suspensionseparated from the milling media was mixed in a product vessel withpost-milling diluent containing about 0.5-3% glycerin, about 0.1-1%sodium chloride, about 0.001-0.1% EDTA, and about 0.001-0.05% BAC. Thismethod produced a composition including about 0.1-2% loteprednoletabonate, about 0.01-2% Pluronic® F127, about 0.5-3% glycerin, about0.1-1% sodium chloride, about 0.001-0.1% EDTA, and about 0.001-0.05%BAC.

Example 12

The following describes a non-limiting example of the effect ofPluronic® F127 on the mucus penetrating properties of the formulations.

The formulations were formed using methods similar to the methoddescribed in Example 10. FIG. 21 shows mass-transport-into-mucus data ofthe following formulations: particles comprising loteprednol etabonateand Pluronic® F127 (LE F127), particles comprising loteprednol etabonateand sodium dodecyl sulfate but not Pluronic® F127 (LE SDS), and marketedformulation Lotemax®. The ratio of loteprednol etabonate to Pluronic®F127 1:1 wt % and the ratio of loteprednol etabonate to SDS is 50:1 wt%. The mass transport was measured according to the procedure describedin Example 2. The results shown in FIG. 21 indicate that the mucuspenetrating properties of LE F127 were approximately 20 times greatercompared to LE SDS and approximately 40 times greater compared toLotemax®. It is believed that an ophthalmic formulation of a drugincluding a loteprednol etabonate core and a coating of F127 may enhancethe ocular exposure of the drug.

Example 13

This non-limiting example shows that loteprednol etabonate (LE) MPPs maybe gamma-irradiated for terminal sterilization without adverselyaffecting the LE MPPs' particle stability, chemical stability, andpharmacokinetics and that glycerin gives chemical protection to the LEMPPs against gamma irradiation.

Gamma irradiation of a formulation can cause chemical degradation andfree radical generation and is a concern especially in aqueousformulations. LE is a soft steroid designed to metabolize via thehydrolytic or enzymatic cleavage of two ester bonds into PJ-91 andPJ-90. When LE is exposed to gamma irradiation,17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-4-ene-17-carboxylicacid chloromethyl ester (tetradeca) and17α-[(ethoxycarbonyl)oxy]-3,11-dioxoandrosta-1,4-diene-17-carboxylicacid chloromethyl ester (11-keto) are formed. When LE isgamma-irradiated, 11-keto appears in small amounts, whereas the amountof tetradeca rapidly increases to above 1%.

The formulations comprising LE and different concentrations of glycerinwere formed using methods similar to the methods described in Examples10-11. Table 13 shows the concentrations of certain degradants of LEafter the formulations were exposed to gamma radiation. Theconcentrations of the degradants were measured immediately after beingsubjected to gamma irradiation (“Gamma irradiated initial”) and 4 weeksafter being subjected to gamma irradiation (“gamma irradiated after 4weeks”) using a Cobalt 60 gamma irradiation source at a dose of 25 kGy.In LE MPPs with no glycerin, the amount of tetradeca increased by18-fold after the gamma irradiation of the LE MPPs. In contrast, aftergamma irradiation little tetradeca was formed in the LE MPPs with 1.2%or 2.4% glycerin. Surprisingly, the levels of PJ-91 and PJ-90 weredecreased in all cases. Although 11-keto was observed after the gammairradiation, the level of 11-keto did not exceed 0.2%. The results showthat much less tetradeca is produced upon gamma radiation when glycerinis present in the formulations. Different concentrations of glycerinwere used (e.g., 1.2 wt % and 2.4 wt %), and the resulting formulationsgenerated similar levels of tetradeca after exposure to gamma radiation.These results are unexpected and suggest that glycerin, commonly used asa tonicity agent, may give chemical protection to the formulationsduring gamma radiation.

TABLE 13 Concentrations of LE and certain degradants of LE after LEformulations were exposed to 25 kGy of gamma radiation.* FormulationConcentration (wt %) composition Time of gamma radiation PJ-90 PJ-91 LETetradeca 11-keto LE, F127, NaCl, Not irradiated 0.48 0.97 97.22 0.080.04 EDTA, BAC Gamma irradiated initial 0.07 0.45 96.51 1.45 0.13 Gammairradiated after 4 weeks 0.07 0.46 96.55 1.49 0.16 LE, F127, 2.4% Notirradiated 0.48 0.87 97.21 0.08 0.05 Glycerin, EDTA, Gamma irradiatedinitial 0.31 0.70 97.28 0.10 0.15 BAC Gamma irradiated after 4 weeks0.36 0.81 97.24 0.11 0.16 LE, F127, 1.2% Not irradiated 0.38 0.78 97.930.06 0.09 Glycerin, NaCl, Gamma irradiated initial 0.29 0.48 98.04 0.090.16 EDTA, BAC Gamma irradiated after 2 weeks 0.28 0.50 97.80 0.15 0.20*LE MPPs employed in this Example were lab scale batches and may be lesspure than batches used in other Examples described herein.

In addition to the chemical stability of a particle formulation, thephysical stability of the formulation is also of high importance. Asshown in FIG. 33A, LE MPP formulations containing sodium chloride andglycerin showed no change in particle size over a month after exposureto 25 kGy of gamma irradiation.

The effects of gamma irradiation on the pharmacokinetics (PK) of LE MPPswas also studied to investigate whether the performance of the LE MPPswas unaffected by gamma irradiation. In vivo, a single topicalinstillation of LE MPPs, which had been gamma irradiated at 25 kGy(Formulation 1 GAMMA, Formulation 2 GAMMA), to New Zealand white rabbitsproduced the same LE levels in the cornea of the rabbits as did LE MPPsthat had not been exposed to gamma irradiation (Formulation 1,Formulation 2; FIG. 33B). In FIG. 33B, Formulation 1 (and Formulation 1GAMMA) included a higher level of Pluronic® F127 than Formulation 2 (andFormulation 2 GAMMA). These results demonstrate that LE MPPs can beterminally sterilized by gamma irradiation without adverse effects onthe particle stability, API (active pharmaceutical ingredient) chemicalstability, or PK of the LE MPPs.

Example 14

This non-limiting example shows that NaCl is beneficial to the stabilityof an LE MPP formulation described herein during dilution of theformulation with water.

The formulations comprising LE and one or more tonicity agents (e.g.,NaCl, Tyloxapol, glycerin, and SSC (an aqueous solution of sodiumcitrate and about 1% NaCl)) were formed using methods similar to themethod described in Examples 10-11. Table 14 shows the particle size andpolydispersity index (PDI) of the LE formulations measured by DynamicLight Scattering (DLS) before and after a 10-fold dilution of theformulations with water. In entries 1, 4, and 5, where the formulationsdo not include NaCl, the particle size (measured by diameter) increasedby about 2-3 fold upon dilution of the formulation with water, mostlikely due to aggregation of the particles. In entries 4 and 5, the PDIalso increased by about 2-3 fold. In contrast, in entries 2, 3, 6, and7, where the formulations included NaCl, the particle size remainedrelatively constant upon dilution of the formulation with water. Inentries 2, 3, and 7, there was also no significant increase in PDI.These results were unexpected because the addition of NaCl (a tonicityagent) to a particle formulation increases the ionic strength of theformulation, and is generally known to destabilize the particleformulation by causing aggregation of particles. The opposite effect wasobserved here.

TABLE 14 Particle size and polydispersity index (PDI) of LE formulationsmeasured by Dynamic Light Scattering (DLS) before and after a 10-folddilution with water. Before dilution After dilution Diameter DiameterEntry Formulation composition (nm) PDI (nm) PDI 1 1-5% LE, 1-5% F127 2080.148 395 0.156 2 1-5% LE, 1-5% F127, 0.9% NaCl, 188 0.102 187 0.1220.05% EDTA, 0.01% BAC 3 1-5% LE, 1-5% F127, 0.9% NaCl, 190 0.128 1930.139 0.1% Tyloxapol, 0.05% EDTA, 0.01% BAC 4 1-5% LE, 1-5% F127, 2.4%Glycerin, 197 0.133 514 0.409 0.05% EDTA, 0.01% BAC 5 1-5% LE, 1-5%F127, 2.4% Glycerin, 191 0.116 389 0.337 0.1% Tyloxapol, 0.05% EDTA,0.01% BAC 6 1-5% LE, 1-5% F127, SSC, 212 0.096 244 0.176 0.05% EDTA,0.01% BAC 7 1-5% LE, 1-5% F127, SSC, 0.1% 212 0.100 246 0.095 Tyloxapol,0.05% EDTA, 0.01% BAC

Example 15

This non-limiting example shows that LE MPPs resulted in increasedexposure when administered topically to an eye, compared to similarlysized non-MPPs.

LE MPPs were compared to LE SDS particles, similarly sized non-MPPs(Table 15). LE SDS particles are produced using methods similar to themethods described in Examples 10-11, except that the particles arecoated with sodium dodecyl sulfate (SDS). Conventional particles, suchas those coated in SDS, are extensively trapped by the peripheralrapidly-cleared mucus layer in the eye and are rapidly cleared. LE MPPsare able to avoid adhesion to, and effectively penetrate through, mucusto facilitate sustained drug release directly to underlying tissues. Invivo, a single topical instillation of LE MPPs to New Zealand whiterabbits produced a 4.4-fold enhancement in the AUC of LE concentrationin the cornea, compared to an equivalent dose of LE SDS, despite bothbeing similarly sized nanoparticles (FIG. 23A). Additionally, despitethe different particle sizes of LE SDS and Lotemax®, which is acommercially available microparticle, the concentrations of LE obtainedfrom rabbits dosed with LE SDS were statistically equivalent to theconcentrations of LE obtained from rabbits dosed with Lotemax® (FIG.23B). These results demonstrate that the MPP's capability to enhance theexposure, in the eye, of a drug that is formulated with the MPP is notsolely due to the small particle size of the MPP.

TABLE 15 Particle size and polydispersity index (PDI) of LE SDS and LEMPP measured by Dynamic Light Scattering (DLS). Diameter Sample (nm) PDILE SDS 240 0.143 LE MPP 241 0.096

Example 16

This non-limiting example shows that LE MPPs resulted in increasedexposure when administered topically to an eye, compared to Lotemax®coated with Pluronic® F127.

LE MPPs were compared to Lotemax®+F127, a formulation in which F127 (0.5wt %) was added to Lotemax®. In vivo, a single topical instillation ofLE MPPs to New Zealand white rabbits produced significantly higherexposure of LE in cornea, compared to an equivalent dose of Lotemax® orLotemax®+F127 (FIG. 24). LE MPPs result in a4.4- and 2.3-foldenhancement of AUC of LE concentration in cornea over Lotemax® andLotemax®+F127, respectively. While Lotemax®+F127 does result a 2-foldincrease of AUC of LE concentration in cornea as compared to Lotemax®alone, these results demonstrate that the MPP's capability to enhancethe exposure, in the eye, of a drug that is formulated with the MPP isnot solely due to the presence of Pluronic® F127 in the formulation.

Example 17

This non-limiting example shows that a formulation including LE MPPenhances exposure of LE in the anterior chamber of the eye compared toLotemax®.

In order to demonstrate that the enhanced exposure of LE from LE MPPstranslates not only to the surface of the eye, but penetrates within theocular globe, levels of LE in the aqueous humor obtained from LE MPPsand Lotemax® formulations were compared. In vivo, a single topicalinstillation of LE MPPs to New Zealand white rabbits producedsignificantly higher LE levels in the aqueous humor compared to a doseof Lotemax® despite the fact that the Lotemax® dose is 20% larger (FIG.25). LE MPPs (containing 0.4% LE) result in an AUC_(0-3hr), enhancementof 3 fold over Lotemax® (containing 0.5% LE). These results demonstratethe enhanced exposure achievable with the MPP technology is not limitedto the surface of the eye but extends into the anterior chamber. Inaddition, the dose of LE can be lowered by 20% with the LE MPPformulation and can still achieve enhanced exposure compared toLotemax®.

Example 18

This non-limiting example demonstrates that a formulation including LEMPP that contains 20% less LE showed improved exposure in rabbit eye andplasma compared to Lotemax®.

In order to demonstrate the enhanced exposure from LE MPP can besustained at lower doses than currently marketed formulations, such asLotemax®, LE MPP was given at a dose 20% lower than Lotemax®. Levels ofLE and two of its main metabolites, PJ-91 and PJ-90, from LE MPP andLotemax® were determined. In vivo, a single topical instillation of LEMPP containing 0.4 wt % LE to New Zealand white rabbits producedsignificantly higher levels of LE in all tissues/fluids tested (e.g.,conjunctiva, cornea, aqueous humor, iris and ciliary body (ICB), centralretina, and plasma) compared to a dose of Lotemax® containing 0.5 wt %LE, despite the fact that the Lotemax® dose is 20% larger than the LEMPP dose (FIGS. 26A-26R). Pharmacokinetic parameters are listed in Table16. These results demonstrate the dose of LE can be lowered by 20% withthe LE MPP formulation and still achieve enhanced exposure overLotemax®.

TABLE 16 Pharmacokinetic parameters of Loteprednol Etabonate (LE) inocular tissues in vivo. Rabbits were given one 50 μL dose of 0.5%Lotemax ® or 0.4% LE MPP in each eye. Aqueous Iris Ciliary Retina PlasmaHumor Conjunctiva Cornea Body (ICB) (center-punch) Lotemax ® T_(max) (h)0.25 0.50 0.083 0.083 0.50 0.50 C_(max) (nM) 0.704 14.0 2430 1330 1054.47 t_(1/2 elimination) (h) 1.88 2.31 4.26 3.75 3.04 9.18 AUC_(0-last)(nM · h) 1.62 30.0 3450 2420 194 14.8 AUC_(0-inf) (nM · h) 1.69 30.54070 2710 200 22.4 LE MPP T_(max) (h) 0.50 0.50 0.083 0.083 0.25 0.50C_(max) (nM) 2.55 43.7 6280 4850 293 14.5 t_(1/2 elimination) (h) 1.711.57 1.92 1.89 1.49 1.55 AUC_(0-last) (nM · h) 4.55 66.0 3450 3570 44231.5 AUC_(0-inf) (nM · h) 4.71 66.4 3490 3610 445 31.9

Example 19

This non-limiting example demonstrates the fluticasone release profileof fluticasone-loaded MPPs containing a PEGylated copolymer and anon-PEGylated core-forming polymer.

Fluticasone-loaded MPPs were prepared according to methods similar tothe ones described herein (e.g., methods described in Example 21) byco-precipitating fluticasone with PLA7A (Surmodics 100DLA7A, MW=108 KDa)as the main polymer and a PEGylated copolymer (e.g., 100DL9K-PEG2K or8515PLGA54K-PEG2K) as the secondary polymer. The ratios of the polymericcomponents tested were 10/90, 20/80, and 30/70 (with PLA7A being themain component in all cases). Various PEGylated copolymers were testedin order to explore the impact of the block composition (i.e., MW of thePEG block vs. MW of the hydrophobic block) on properties of resultingparticles. In particular, the ability of resulting particles topenetrate mucus, control drug release, and maintain colloidal stabilitythroughout the formulation process was assessed.

It was found that, while many compositions led to satisfactory drugrelease and mucus-penetration, good colloidal stability could not beachieved with most of them (see FIG. 27). Colloidal stability isespecially important since the current formulation process involvesisolation and purification of the MPPs by centrifugation andresuspension. The poor colloidal stability of many compositions wasmanifested, in part, by the inability to resuspend product obtainedafter the centrifugation step. The compositions that resulted in MPPswith good colloidal stability as well as good control over drug release(e.g., a continuous release over 24 hours in vitro as in this Example)appear to have a relatively high surface coverage of PEG on the particle(e.g., at least about 0.18 PEG chains per nm²) at a relatively lowoverall PEG content in the particle (e.g., less than about 3 wt % of thetotal polymer content). In other words, combinations of PLA7A withPEG-copolymers with a relatively short hydrophobic block (e.g.,100DL9K-PEG2K) result in MPPs that are more colloidally stable thansimilar combinations of PEG-copolymers with a relatively longhydrophobic block (e.g., 8515PLGA54K-PEG2K) (see FIG. 27).

Example 20

This non-limiting example demonstrates that MPPs comprising sorafenibavoided entrapment in human cervicovaginal mucus and were able todiffuse through mucus.

MPPs comprising sorafenib may be prepared according to methods describedherein (e.g., methods described in Examples 21 and 29). Mobility ofconventional nanoparticles and the MPPs described herein in humancervicovaginal mucus was characterized by bulk transport and/ormicroscopy as described in Examples 2 and 10, respectively. The resultsare shown in FIGS. 28A-28B. The conventional nanoparticles were trappedin human cervicovaginal mucus, whereas the MPPs described herein avoidedentrapment and were able to diffuse through mucus.

Example 21

This non-limiting example demonstrates that topical delivery ofsorafenib, (a small molecule receptor tyrosine kinase (RTK) inhibitor)formulated as MPP greatly enhances sorafenib levels in the retina andchoroid of the eye. This example also shows that sorafenib levels inanterior segment tissues of the eye depend on the MPP release rate andcan be reduced without significantly affecting sorafenib levels at theback of the eye.

Sorafenib-loaded MPPs with relatively fast drug release (MPP1) wereprepared by a milling procedure: an aqueous dispersion containing drugand Pluronic F127 (F127) was stirred with zirconium oxide beads, asgrinding medium, until particle size was reduced below 300 nm asmeasured by dynamic light scattering. This method employs excipientsapproved by the FDA for use in ophthalmic products and produced a stableaqueous nanosuspension of MPPs.

Sorafenib-loaded MPPs with relatively slow drug release kinetics (MPP2)were prepared by encapsulating sorafenib into a biodegradable polymericnanoparticle decorated with a coating described herein. For example, asolution containing sorafenib free base (LC Labs), PLA (Polylactide,100DL7A, Surmodics), and PLA-PEG (poly(ethylene glycol)-co-polylactide,100DL-mPEG2K, Surmodics) in tetrahydrofuran was added at a controlledrate to an excess of aqueous solution of Pluronic® F127 with stirring.The produced particles were stirred at room temperature to let thevolatiles evaporate and to crystallize out sorafenib that wasunencapsulated. The crystals of unencapsulated sorafenib were removed byfiltration through a suitable sized glass fiber filter. Thenanoparticles were isolated from the filtrate by centrifugation andwashed once with aqueous Pluronic® F127. The final product ofnanoparticles was resuspended in Pluronic® F127.

Sorafenib concentrations in the MPP formulations were confirmed by HPLC.The size of the MPPs was measured by dynamic light scattering, using aZetasizer Nano ZS90 (Malvern Instruments). In vitro drug release wasevaluated at 37° C. in 50 mM phosphate buffer (pH 7.4) in the presenceof 0.5% Tween 80 to ensure sink conditions, such as experimentalconditions that are well-below saturation solubility.

The pharmacokinetics of sorafenib following a single topicaladministration of the MPPs or a non-MPP comparator was evaluated in NewZealand white rabbits (NZW) at a contract research organization. Anaqueous suspension of sorafenib was used as the non-MPP comparator. Eachanimal received a 50 μL topical instillation containing 5 mg/mLsorafenib in both eyes (n=6). Ocular tissues, including cornea and an 8mm punch of choroid and of retina from the back of the eye, wereharvested at various time points. The punch was used to target the areaat the back of the eye where the human macula would be as this is thetarget for AMD therapy. Sorafenib levels were determined by LC/MS.

MPP1 and MPP2 formulated as described above formed stablenanosuspensions with a Z-average diameter of 187 nm (PDI=0.172) and 222nm (PDI=0.058), respectively. Since MPP1 was essentially a suspension ofthe pure drug sorafenib, the drug release was driven largely by drugdissolution, which is relatively rapid. In the case of MPP2, the drugsorafenib was encapsulated in PLA polymers, and the drug loading was20%. The polymeric composition of MPP2, including the molecular weightof PLA, the ratio of PLA to PLA-PEG, and the composition of PLA-PEG, wassystematically varied in order to achieve a release ratewell-differentiated from MPP1. The MPP2 formulation demonstratedcontinuous drug release over about 24 hrs in vitro.

In the cornea, a single dose of the fast-releasing MPP1 formulationproduced sorafenib levels up to 18-fold higher than those from thecomparator and sustained an at least 7-fold enhancement over thecomparator for at least 6 hours. In contrast, the slow-releasing MPP2formulation produced only an approximately 3-fold enhancement over thecomparator steadily sustained over the course of 6 hours. However, inposterior segment tissues (e.g., retina and choroid), both MPP1 and MPP2produced similarly high sorafenib levels, well-outperforming thecomparator (FIGS. 29A-29B). In fact, the levels of sorafenib in theretina produced by the MPP formulations approached or exceeded reportedcellular IC₅₀ values against VEGFR-2 (37 ng/g) and PDGFR-β (14 ng/g) forsorafenib, a relatively low potency, first-generation RTK inhibitor.Furthermore, both MPP formulations were well tolerated as assessed byDraize scoring.

These results not only demonstrate a proof-of-concept that the MPPsdescribed herein, and compositions thereof, can greatly enhance deliveryof a pharmaceutical agent to the back of the eye via topicaladministration but also suggest that topical delivery of a smallmolecule RTK inhibitor formulated as MPPs may have a potential in thetreatment of a broad range of ocular diseases, such as AMD.

Example 22

This non-limiting example demonstrates that a formulation including LEMPPs improved the exposure of LE in the aqueous humor of the rabbit eyecompared to Lotemax® gel.

In order to demonstrate that enhanced exposure of LE from LE MPPs can besustained at lower doses compared not only to marketed suspensionformulations, but also to marketed gel formulations, levels of LE weredetermined when LE MPPs (dosed at 0.4% LE) and Lotemax® gel (dosed at0.5% LE) were used. Gel and ointment formulations are popularly used inan attempt to increase exposure in the eye by delivering LE in a viscousmatrix. Gel and ointment formulations often blur vision and are lesscomfortable and harder to install than a liquid eye drop.

To generate LE MPPs, a milling procedure was employed according to themethods described herein. For example, an aqueous dispersion containingLE and Pluronic® F127 (F127) was milled with a grinding medium until theparticle size was reduced to below about 300 nm as measured by dynamiclight scattering. Mucus mobility was characterized in humancervicovaginal mucus based on the previously described characterizationmethods.

In vivo, a single topical instillation of LE MPPs (KPI-121) to NewZealand white rabbits produced higher LE levels in aqueous humorcompared to a dose of Lotemax® gel despite the fact that the Lotemax®gel dose was 20% higher and in a viscous matrix (FIG. 30). The AUC₀₋₃ ofthe LE MPPs was 1.5 times higher than that of Lotemax® gel. The C_(max)of the LE MPPs was 2.4 times higher than that of Lotemax® gel. Theseresults indicate that the MPPs described herein outperform the viscousmatrix used in Lotemax® gel and that the dose of LE may be lowered by20% with the LE MPP formulation and may still achieve similar orenhanced exposure compared to Lotemax® gel.

Example 23

This non-limiting example demonstrates that LE MPPs show dose-dependentexposure in the aqueous humor of New Zealand white rabbits.

In order to demonstrate that the exposure of LE from an LE MPPformulation is dose dependent, a dose ranging study was performed. Togenerate LE MPPs, a milling process according to the methods describedherein was employed. For example, an aqueous dispersion containing drugand Pluronic® F127 (F127) was milled with a grinding medium until theparticle size was reduced to below about 300 nm as measured by dynamiclight scattering. Mucus mobility was characterized in humancervicovaginal mucus based on the previously described characterizationmethods. Dilutions were performed to give LE MPP suspensions thatincluded 0.4%, 0.5%, 0.6%, or 1% LE. In vivo, a single topicalinstillation of LE MPPs to New Zealand white rabbits produced LE levelsin the aqueous humor of the rabbits which were dependent on the dosegiven (FIGS. 31A-31B). These results demonstrate the LE MPPs exhibitdose-dependent pharmacokinetics (PK).

Example 24

This non-limiting example demonstrates that LE MPPs may be stablyformulated in the presence of one or more ionic components, such assodium chloride.

In order to demonstrate that LE MPPs may be formulated with ioniccomponents, such as ionic salts, and can remain physically stable insuch a formulation, sodium chloride, a tonicity agent with a well-knownsafety profiles in humans, was introduced into the LE MPPs. It iscommonly known in the art that ionic components should not be added toparticle suspensions as the ionic components tend to destabilize theparticle suspensions. Surprisingly, this is not the case with LE MPPs.

It is known that, to achieve a formulation of an osmolarity of about 300mOsm/kg, the concentration of sodium chloride in the formulation istypically about 0.9%. A combination of 1.2% glycerin and 0.45% sodiumchloride generally also yields an isotonic solution and was tested tocompare different levels of sodium chloride.

To generate nanoparticles, a milling process according to the methodsdescribed herein was employed. For example, an aqueous dispersioncontaining LE and Pluronic® F127 (F127) was milled with a grindingmedium until the particle size was reduced to below about 300 nm asmeasured by dynamic light scattering. The physical stability of theresulted isotonic formulations was tested by monitoring the size of theparticles in the formulations using dynamic light scattering (DLS). Itwas discovered that the particles in the formulations containing twodifferent concentrations of sodium chloride were very stable in size asshown in FIG. 32. In FIG. 32, the data depicted with the triangularmarkers had a higher percentage of NaCl in the formulation than the datadepicted with the circular markers. These results demonstrate that LEMPPs can be formulated into stable compositions in the present of ioniccomponents, such as sodium chloride.

Example 25

This non-limiting example demonstrates that particles containingPluronic® F127 and diclofenac or ketorolac may be mucus penetrating.

In order to demonstrate that particles comprising a core containing anNSAID and comprising a surface-altering agent (e.g., Pluronic® F127) maybe mucus penetrating, two NSAIDs, i.e., diclofenac and ketorolac, werestudied.

To form particles containing diclofenac or ketorolac, a millingprocedure was employed according to methods described herein. In one setof experiments, an aqueous dispersion containing Pluronic® F127 and oneof ketorolac free acid and diclofenac free acid was milled with agrinding medium until the particle size was reduced to below about 300nm as measured by dynamic light scattering. To generate similarly sizednon-MPP comparators, a similar milling procedure was employed exceptthat SDS was used as the surface-altering agent instead of Pluronic®F127. The mucus mobility of the produced particles was characterized inhuman cervicovaginal mucus based on the previously described microscopymethods. In the case of diclofenac, the mucus mobility was additionallycharacterized by the previously described bulk transport method. Theresults are shown in FIG. 41. The data demonstrate that the particlescontaining Pluronic® F127 and one of ketorolac and diclofenac were mucuspenetrating, whereas the particles containing SDS and one of ketorolacand diclofenac were not mucus penetrating.

Example 26

This non-limiting example demonstrates a method of forming MPPsincluding bromfenac calcium, and compositions and/or formulationsthereof.

MPPs including bromfenac calcium as the core and Pluronic® F127 (F127)as a muco-inert surface-altering agent, and compositions and/orformulations including these MPPs, were prepared using a method similarto the methods described in Example 2. In one set of experiments,bromfenac calcium was nanomilled in an aqueous dispersion containingbromfenac calcium and Pluronic® F127 using zirconium oxide beads asgrinding medium, until the particle size was reduced below 300 nm asmeasured by dynamic light scattering. The resulted nanomilled suspensioncan be diluted to a lower concentration if desired. In some experiments,bromfenac calcium was milled in water, 125 mM of CaCl₂, or 50 mM of Trisbuffer to give three formulations. The particle size of the obtainedMPPs in the three formulations was measured by dynamic light scattering,and the results are shown in FIG. 34. All three formulations had aZ-average diameter of about 200 nm and a polydispersity index <0.2.These data demonstrate that the bromfenac calcium MPPs are small anduniform in size and thus are suitable for ocular applications.

Example 27

This non-limiting example demonstrates that MPPs including bromfenaccalcium, and compositions and/or formulations thereof, are stable whenstored at room temperature.

MPPs including bromfenac calcium, and compositions and/or formulationsthereof, may be prepared according to methods in Example 26. The MPPs,compositions, and/or formulations were stored at room temperature fordays, and the particle Z-average size and polydispersity index of theMPPs were determined by dynamic light scattering. The results are shownin FIGS. 35A-35D. These data demonstrate that the bromfenac calcium MPPsmaintained good particle size stability over extended period of timewhen stored at room temperature.

Enhanced mucus mobility of bromfenac calcium MPPs in humancervicovaginal mucus was confirmed by fluorescent microscopy and highresolution dark field microscopy (data not shown).

Example 28

This non-limiting example demonstrates that excipients in compositionsand/or formulations containing bromfenac calcium MPPs may improve thechemical stability of bromfenac calcium MPPs.

To improve the chemical stability of bromfenac calcium in MPPs,different excipient compositions were explored for the milling step andthe final formulation that either (1) lower the solubility of bromfenacor (2) maintain a pH range at which bromfenac is most stable. Table 20shows the pH and solubility of bromfenac calcium MPPs in two buffers(125 mM CaCl₂ and 50 mM Tris) and, for comparison, in unbuffered water.The chemical stability of bromfenac calcium MPPs in 125 mM CaCl₂, whichreduced the drug solubility, and in 50 mM Tris, which maintained the pHof the solution at about 8, was markedly enhanced compared to that inwater.

TABLE 20 pH, solubility, and chemical stability of bromfenac calcium MPPsuspensions containing 0.09% w/v bromfenac in the presence of variousexcipient compositions at room temperature.* Formulation: bromfenaccalcium suspension (0.09% w/v bromfenac) Vehicle Concentration ofFormulation Properties Pluronic ®F127 Solubility Buffer (% w/v) pH(mg/mL) % peak area* None 0.5 6.87 0.21 0.13 (day 7)   (water) 0.09 6.750.20 0.09 (day 23)  125 mM 0.5 6.48 0.03 0 (day 7) CaCl₂ 0.09 6.60 0.07 0 (day 23) 50 mM 0.5 8.03 0.25 0 (day 7) Tris 0.09 7.98 0.27  0 (day23) buffer *The chemical stability of bromfenaccalcium was determined as% chromatographic peak area for the lactam degradant of bromfenac.

Example 29

This non-limiting example demonstrates that MPPs containing sorafenib orlinifanib enhanced the exposure of sorafenib or linifanib at the back ofthe eye of rabbits.

In order to demonstrate that enhanced mucus penetration is useful notonly at the front of the eye, but can lead to enhanced exposure at theback of the eye as well, sorafenib and linifanib, two receptor tyrosinekinase inhibitors, were formulated as MPPs. Small molecule RTKinhibitors that act on vascular endothelial growth factor receptor(VEGFR) have potential as therapy for age-related macular degeneration(AMD). If topical delivery of an RTK inhibitor could provide sufficientlevels of the RTK inhibitor at the back of the eye then repeatedintravitreal injections, used by current therapies, could be avoided.Delivery of an RTK inhibitor to the back of the eye would also bebeneficial in a number of other diseases which affect posterior segmenttissues.

To generate MPPs containing an RTK inhibitor (e.g., sorafenib andlinifanib), a milling procedure similar to that used for LoteprednolEtabonate was employed: an aqueous dispersion containing the RTKinhibitor and F127 or sodium dodecyl sulfate (SDS) was milled with agrinding medium until the particle size was reduced to below about 300nm as measured by dynamic light scattering. Mucus mobility wascharacterized in human cervicovaginal mucus based on the previouslydescribed characterization methods. Sorafenib and linifanib wereprocessed into MPPs when milled with F127 and into non-MPPs when milledwith SDS.

In vivo, a single 50 μL topical instillation of 0.5% sorafenib-MPP toNew Zealand White rabbits produced sorafenib levels in the retina of therabbit which were 45 times higher than sorafenib levels from the non-MPPcontrol at 2 hours and 96 fold above cellular IC₅₀ (FIG. 36A). A centralpunch of 8 mm in diameter was taken from the retina where the humanmacula would be in order to measure sorafenib levels at the back of theeye where AMD therapies would be targeted. The sorafenib-MPP outperformsthe sorafenib non-MPP by a statistically significant amount.

In vivo, a single 50 μL topical instillation of 2% linifanib-MPP to NewZealand White rabbits produced linifanib levels in the center punchretina which were approximately 2 fold higher than linifanib levels fromthe non-MPP control over the full 4 hours examined and 777 fold abovecellular IC₅₀ at 4 hours (FIG. 36B). Again the difference between MPPand non-MPP was statistically significant.

These results demonstrate that enhanced mucus penetration can enablehigher drug exposure at the back of the eye. The application of thistechnology can be used to improve drug exposure in any tissue of theeye, whether at the ocular surface or the back of the eye.

Example 30

This non-limiting example demonstrates that MPPs containing MGCD-265 orpazopanib generated therapeutically relevant levels of MGCD-265 orpazopanib at the back of the eye of rabbits.

In order to demonstrate the wide applicability of the MPP technology tothe delivery of small molecule RTK inhibitors in therapeuticallyrelevant (e.g., therapeutically effective) levels to the back of theeye, two additional compounds were studied: MGCD-265 and pazopanib.

To generate the MPPs, a milling procedure similar to that used forloteprednol etabonate was employed: an aqueous dispersion containingMGCD-265 or pazopanib and F127 was milled with a grinding medium untilthe particle size was reduced to below about 300 nm as measured bydynamic light scattering. Mucus mobility was characterized in humancervicovaginal mucus based on the previously described characterizationmethods. Both MGCD-265 and pazopanib were processed into MPPs whenmilled with F127.

In vivo, a single 50 μL topical instillation of 0.5% pazopanib-MPP toNew Zealand White rabbits produced pazopanib levels in the center punchretina which were 9 fold higher than the cellular IC₅₀ at 4 hours (FIG.37A).

In vivo, a single 50 μL topical instillation of 2% MGCD-265-MPP to NewZealand White rabbits produced MGCD-265 levels in the retina which were37 fold higher than the cellular IC₅₀ at 30 minutes and 116 fold higherthan the cellular IC₅₀ at 4 hours (FIG. 37B).

These results, along with Example 29, demonstrate that a variety of RTKinhibitors can be formulated as MPPs and the levels of the RTKinhibitors achieved at the back of the eye with topical administrationare relevant to the active concentrations (cellular IC₅₀) of the RTKinhibitors.

Example 31

This non-limiting example demonstrates that a single topicaladministration of cediranib-MPPs produced therapeutically relevant druglevels at the back of the eye of rabbits for 24 hours.

In order to demonstrate the potential for MPPs containing a drug tosustain therapeutically relevant drug levels at the back of the eye ofrabbits over a 24 hour period, cediranib, an RTK inhibitor, wasformulated as MPPs. If topical delivery of a drug could providetherapeutically relevant levels of the drug at the back of the eye,repeated intravitreal injections, which are used in current AMDtherapies, could be avoided. Ideally, this topical therapy would sustaindrug levels at the back of the eye to provide less frequent dosing.

To generate the MPPs, a milling procedure similar to that used forLoteprednol Etabonate was employed: an aqueous dispersion containingcediranib and Pluronic® F127 was milled with a grinding medium until theparticle size was reduced to below about 300 nm as measured by dynamiclight scattering. Mucus mobility was characterized in humancervicovaginal mucus based on the previously described characterizationmethods. Cediranib was processed into MPPs when milled with F127.

In vivo, a single 50 μL topical instillation of 2% cediranib-MPP toHY79b pigmented rabbits produced cediranib levels in the choroid of therabbit which were 4,800 fold over cellular IC₅₀ at 24 hours (FIG. 38A)and cediranib levels in the retina of the rabbit which were 1,000 foldover cellular IC₅₀ at 24 hours (FIG. 38B). These results demonstrate theexposure achievable at the back of the eye with the MPP technology canbe in therapeutically relevant ranges and that relevant drug exposurecan be maintained over a long period of time (e.g., at least 24 hours).

Example 32

This non-limiting example demonstrates that a single topicaladministration of axitinib-MPP produced therapeutically relevantaxitinib levels at the back of the eye of Dutch belted rabbits for 24hours.

In order to demonstrate the potential for MPPs to sustaintherapeutically relevant drug levels at the back of the eye of rabbitsover a 24 hour period, axitinib, an RTK inhibitor, was formulated asMPPs. If topical delivery of a drug could provide therapeuticallyrelevant levels of the drug at the back of the eye, repeatedintravitreal injections, which are used by current AMD therapies, couldbe avoided. Ideally, this topical therapy would sustain drug levels atthe back of the eye to provide less frequent dosing.

To generate nanoparticles, a milling procedure similar to that used forloteprednol etabonate was employed: an aqueous dispersion containingaxitinib and F127 was milled with a grinding medium until the particlesize was reduced to below about 300 nm as measured by dynamic lightscattering. Mucus mobility was characterized in human cervicovaginalmucus based on the previously described characterization methods.Axitinib was processed into MPPs when milled with F127.

In vivo, a single 50 μL topical instillation of 2% axitinib-MPP to Dutchbelted rabbits produced therapeutically relevant drug levels in thechoroid of the rabbit 1,100 fold over cellular IC₅₀ at 24 hours (FIG.39A) and levels in the retina of the rabbit which were 37 fold overcellular IC₅₀ at 24 hours (FIG. 39B). These results demonstrate theexposure achievable at the back of the eye with the MPP technology canbe in therapeutically relevant ranges and that relevant drug exposurecan be maintained over a long period of time (e.g., at least 24 hours).

Example 33

This non-limiting example demonstrates that axitinib-MPP reducedvascular leakage in a rabbit VEGF (vascular endothelial growth factorreceptor)-challenge model.

In order to demonstrate the therapeutic potential of the MPP technologyin the treatment of an eye disease of the back of the eye, axitinib-MPPswere studied in an acute VEGF-challenge model. In this model, Dutchbelted rabbits were given an intravitreal injection of VEGF to stimulatevascular growth. Fluorescein angiography was used to determine thedegree to which the rabbits developed abnormal vascular and leakage.Representative images from fluorescein angiography after treatment withvehicle, axitinib-MPP, or Avastin® are shown in FIGS. 40A-40C. Rabbitswhich were dosed with vehicle showed a large amount of vessel growth,tortuosity, and leakage. Rabbits treated with Avastin®, commonly usedoff-label to treat AMD in humans and with a different mechanism ofaction than axitinib, showed no change in retinal vasculature. Rabbitstreated with axitinib-MPP showed some vessel growth but significantlyless leakage than the vehicle group. These results demonstrate theexposure achievable at the back of the eye using the MPP technology issufficient to significantly reduce vascular leakage in an acutechallenge model and has potential as an effective therapy in AMD andother back-of-the-eye diseases.

Example 34

This non-limiting example demonstrates that LE MPPs containing Pluronic®F127, Tween 80®, or PVA as the surface-altering agent showed improvedexposure of LE in rabbits compared to Lotemax®.

In order to demonstrate that the LE MPPs' ability to enhance theexposure of LE is not limited to the inclusion of F127 as thesurface-altering agent in the LE MPPs, two additional surface-alteringagents were studied: Tween 80® and polyvinyl alcohol (PVA). Tween 80® isan FDA approved surface-altering agent which consists of PEGylatedsorbitan forming a head group and an alkyl tail. Tween 80® is differentfrom a range of other surface-altering agents (e.g., F127 and PVA) inthat, among other things, it is oligomeric and thus significantly lowerin molecular weight. PVA is an FDA approved polymer produced by, e.g.,partially hydrolyzing polyvinyl acetate, creating a random copolymer ofpolyvinyl acetate and polyvinyl alcohol. PVA is different from a rangeof other surface-altering agents (e.g., F127 and Tween 80®) in that,among other things, it contains no PEG. In Examples 4-6, it is shownthat some PVAs enable mucus penetration while other PVAs do not. Thisdifferentiated mucus penetration behavior may be controlled by themolecular weight and degree of hydrolysis of PVA. Based on the resultsfrom these Examples, a PVA that has a molecular weight of about 2 kDaand is about 75% hydrolyzed was selected for the study of mucuspenetration properties of LE MPPs.

To form the LE MPPs, a milling procedure was employed as describedherein. In one set of experiments, an aqueous dispersion containing LEand one surface-altering agent selected from F127, Tween 80®, and PVA (2kDa, 75% hydrolyzed) was milled with a grinding medium until theparticle size was reduced to below about 300 nm as measured by dynamiclight scattering. Mucus mobility was characterized in humancervicovaginal mucus based on the previously described characterizationmethods. All three LE MPPs (i.e., LE-F127, LE-Tween80, and LE-PVA)showed mucus penetrating properties (FIG. 42).

In vivo, a single topical instillation of each one of the three LE MPPsto New Zealand white rabbits produced LE levels in the cornea of therabbit which were significantly higher than the LE levels from asimilarly administered dose of Lotemax® (FIG. 42). These resultsdemonstrate that the MPPs, compositions, and/or formulations containinga drug enhance exposure of the drug based on the mucus penetratingproperties of the particles.

OTHER EMBODIMENTS

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1. A method of treating a dry eye condition in an eye of a patient inneed thereof, comprising: administering topically to the eye a topicalpharmaceutical composition comprising (a) a plurality of nanoparticlesof loteprednol etabonate; (b) about 0.5% w/v to about 3% w/v glycerin;(c) about 0.1% w/v to about 1% w/v sodium chloride; and (d) poloxamer407; wherein the nanoparticles of loteprednol etabonate arenon-covalently coated with the poloxamer 407 to form coatednanoparticles, and wherein the coated nanoparticles aremucus-penetrating; wherein the loteprednol etabonate is present in thepharmaceutical composition in an amount between about 0.1% w/v and about2% w/v; and wherein the pharmaceutical composition is a topicalsuspension, and wherein the ratio of the total weight of the loteprednoletabonate to the total weight of the poloxamer 407 comprised in thetopical suspension is about 2:1.
 2. The method of claim 1, wherein theloteprednol etabonate is present in the pharmaceutical composition in anamount between about 0.1% w/v and about 1% w/v.
 3. The method of claim2, wherein the loteprednol etabonate is present in the pharmaceuticalcomposition in an amount between about 0.2% w/v and about 0.4% w/v. 4.The method of claim 3, wherein the pharmaceutical composition furthercomprises about 0.001% w/v to about 0.1% w/v disodiumethylenediaminetetraacetic acid.
 5. The method of claim 4, wherein thepharmaceutical composition further comprises about 0.001 to about 0.05%w/v benzalkonium chloride.
 6. The method of claim 1, wherein the coatednanoparticles have a relative velocity of greater than 0.6 in humancervicovaginal mucus.
 7. The method of claim 1, wherein the poloxamer407 coating on the nanoparticles of loteprednol etabonate is at anaverage density of at least 0.05 molecules/nm².
 8. The method of claim7, wherein the average density is at least 0.1 molecules/nm².
 9. Themethod of claim 8, wherein the coated nanoparticles have an average sizeof about 200 nm to about 700 nm.
 10. The method of claim 9, wherein theaverage size of the coated nanoparticles is as measured in Z-averagediameter by dynamic light scattering.
 11. The method of claim 10,wherein the polydispersity index of the composition is less than orequal to about 0.1.
 12. The method of claim 1, wherein thepharmaceutical composition further comprises one or more degradants ofthe loteprednol etabonate, wherein the one or more degradants of theloteprednol etabonate includes17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-4-ene-17-carboxylicacid chloromethyl ester at less than or equal to about 0.5 wt % relativeto the weight of the loteprednol etabonate.
 13. The method of claim 12,wherein the pharmaceutical composition is made sterile via asterilization process comprising gamma irradiation, and wherein the17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-4-ene-17-carboxylicacid chloromethyl ester is at less than or equal to about 0.5 wt %relative to the weight of the loteprednol etabonate four weeks after thegamma irradiation.
 14. A method for treating a dry eye condition in aneye of a patient in need thereof, comprising: administering topically tothe eye a topical pharmaceutical composition comprising: (a) a pluralityof coated nanoparticles, each of the coated nanoparticles comprising:(i) a core particle comprising loteprednol etabonate, and wherein theloteprednol etabonate comprises at least 90 wt % of the core particle;and (ii) a coating on the core particle, the coating comprisingpoloxamer 407 non-covalently adsorbed to the core particle; and (b)about 0.5% w/v to about 3% w/v glycerin; and (c) about 0.1% w/v to about1% w/v sodium chloride; and wherein the pharmaceutical composition is atopical suspension, and wherein the topical suspension comprises about0.1% to 2% w/v loteprednol etabonate and wherein the ratio of the totalweight of the loteprednol etabonate to the total weight of the poloxamer407 comprised in the topical suspension is about 2:1, wherein the coatednanoparticles are mucus-penetrating.
 15. The method of claim 14, whereinthe core particle is substantially free of a polymeric component. 16.The method of claim 14, wherein the loteprednol etabonate is present inthe pharmaceutical composition in an amount between about 0.1% and about1% w/v.
 17. The method of claim 16, wherein the loteprednol etabonate ispresent in the pharmaceutical composition in an amount between about0.2% and about 0.4% w/v.
 18. The method of claim 17, wherein thepharmaceutical composition further comprises about 0.001% w/v to about0.1% w/v disodium ethylenediaminetetraacetic acid.
 19. The method ofclaim 18, wherein the pharmaceutical composition further comprises about0.001 to about 0.05% w/v benzalkonium chloride.
 20. The method of claim14, wherein the coated nanoparticles have a relative velocity of greaterthan 0.6 in human cervicovaginal mucus.
 21. The method of claim 14,wherein the poloxamer 407 coating on the particles of loteprednoletabonate is at an average density of at least 0.05 molecules/nm². 22.The method of claim 21, wherein the average density is at least 0.1molecules/nm².
 23. The method of claim 14, wherein the coatednanoparticles of have an average size of about 200 nm to about 700 nm.24. The method of claim 23, wherein the average size of the coatednanoparticles is as measured in Z-average diameter by dynamic lightscattering.
 25. The method of claim 24, wherein the polydispersity indexof the composition is less than or equal to about 0.1.
 26. The method ofclaim 14, wherein the pharmaceutical composition further comprises oneor more degradants of the loteprednol etabonate, wherein the one or moredegradants of the loteprednol etabonate includes17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-4-ene-17-carboxylicacid chloromethyl ester at less than or equal to about 0.5 wt % relativeto the weight of the loteprednol etabonate.
 27. The method of claim 24,wherein the pharmaceutical composition is made sterile via asterilization process comprising gamma irradiation, and wherein the17α-[(ethoxycarbonyl)oxy]-11β-hydroxy-3-oxoandrosta-4-ene-17-carboxylicacid chloromethyl ester is at less than or equal to about 0.5 wt %relative to the weight of the loteprednol etabonate four weeks after thegamma irradiation.
 28. (canceled)
 29. (canceled)
 30. A method of makinga pharmaceutical composition comprising: (a) a plurality of coatednanoparticles, each of the coated nanoparticles comprising: (i) a coreparticle comprising loteprednol etabonate, and wherein the loteprednoletabonate comprises at least 90 wt % of the core particle; and (ii) acoating on the core particle, the coating comprising poloxamer 407non-covalently adsorbed to the core particle; and (b) about 0.5% w/v toabout 3% w/v glycerin; and (c) about 0.1% w/v to about 1% w/v sodiumchloride; wherein the pharmaceutical composition is a topicalsuspension, and wherein the topical suspension comprises about 0.1% to2% w/v loteprednol etabonate in total and wherein the ratio of the totalweight of the loteprednol etabonate to the total weight of the poloxamer407 comprised in the topical suspension is about 2:1, and wherein thecoated nanoparticles are mucus-penetrating; the method comprising:milling a coarse aqueous suspension containing about 2-20% loteprednoletabonate in the form of coarse or micronized crystals, about 0.2-20% ofpoloxamer 407, about 0.5-3% glycerin, and about 0.1-1% sodium chloride,in the presence of milling media to produce a nanosuspension comprisingcoated nanoparticles of loteprednol etabonate with poloxamer 407 andsized in the range of about 200 nm to about 500 nm; and separating thenanosuspension of coated loteprednol etabonate particles from themilling media.
 31. The method of claim 5, wherein the pharmaceuticalcomposition further comprises a sodium citrate and citric acid buffer.32. The method of claim 19, wherein the pharmaceutical compositionfurther comprises a sodium citrate and citric acid buffer.